Ip receptor antagonists for the treatment of pathological uterine conditions

ABSTRACT

A method of combating a pathological condition of the uterus in a female individual, the method comprising administering to the individual at least one agent that is an antagonist of the IP receptor and/or a PGIS inhibitor. The pathological condition of the uterus is uterine carcinoma, menorrhagia, dysmenorrhoea or an endomenstrual myometrial pathological condition.

The present invention relates to methods of treatment, and in particularmethods of treating uterine pathological conditions.

Pathological conditions of the uterus represent a serious health problemin women, particularly women of the western world. Such pathologicalconditions include uterine cancers, endometrial or myometrialpathological conditions such as endometriosis (endometrial) and fibroids(myometrial), menorrhagia and dysmenorrhoea.

Epithelial cells of the human endometrium are highly vulnerable toneoplastic transformation. In the western world, endometrial carcinomais the most common gynaecologic malignancy. Endometrial cancer can arisefrom several cell types but the glandular epithelium is the most commonprogenitor (adenocarcinomas account for 80-90% of uterine tumours).Endometrial cancer is predominantly a post-menopausal disease whereincidence is uncommon below the age of forty and peaks by about seventyyears of age. The incidence of endometrial cancer has been increasingsteadily in the western world during the last fifty years and this hasbeen attributed largely to increased life expectancy and improveddetection methods (Gordon & Ireland, 1994; Mant & Vessey, 1994).

Endometriosis is the ectopic implantation and growth of endometrium andcan therefore be considered as abnormal growth of cells of theendometrium. Adenomyosis is a form of endometriosis where the ectopicendometrium is implanted in the myometrium.

Menorrhagia is over-abundance of the menstrual discharge. Dysmenorrhoeameans painful menstruation. Menorrhagia and dysmenorrhoea affect manywomen, particularly in the western world, and represent a significanthealth problem. At least one in 20 women in the UK aged between 34 and49 years will consult their general practitioners because of menstrualproblems. These women account for more than one in ten of allgynaecological referrals and cost the NHS in excess of £7 million peryear for medical prescriptions alone.

Perceived abnormal vaginal bleeding is said to account for 70% of the atleast 70,000 hysterectomies performed each year. At present, thetreatments used for menorrhagia include tranexamic acid or mefenamicacid. In severe cases the treatment is hysterectomy (vaginal orabdominal) but this is a major operation with serious morbidity and somerisk of death. A review of treatments for menorrhagia is Stirrat (1999)The Lancet 353, 2175-2176. The development of further and alternativetherapies is desirable.

Cyclooxygenase (COX) enzymes, also called prostaglandin endoperoxidesynthase (PGHS), catalyse the rate limiting step in the conversion ofarachidonic acid to prostaglandin H₂ (PGH₂). In turn PGH₂ serves as asubstrate for specific prostaglandin synthase enzymes that synthesisethe natural prostaglandins. These are named according to theprostaglandin they produce such that prostaglandin D₂ is synthesised byprostaglandin-D-synthase, prostaglandin E₂ (PGE₂) byprostaglandin-E-synthase (PGES), prostaglandin F_(2α) (PGF_(2α)) byprostaglandin-F-synthase (PGFS), and prostacyclin (prostaglandin I₂,PGI₂) by prostaglandin-I-synthase (PGIS).

To date, there are two identified isoforms of the COX enzyme, COX-1 andCOX-2 (DeWitt, 1991). COX-1 is constitutively expressed in many tissuesand cell types and generates prostaglandins for normal physiologicalfunction (Herschman, 1996). By contrast, the expression of COX-2 israpidly induced following stimulation of quiescent cells by growthfactors, oncogenes, carcinogens and tumour-promoting phorbol esters(Herschman, 1996; Subbaramaiah et al, 1996).

Prostacyclin (PGI₂) has been characterised as a vasodilator and a potentinhibitor of platelet aggregation (Smyth & Fitzgerald, 2002) and as suchplays an essential role in the maintenance of vascular haemostasis. Itis the main prostanoid synthesised by vascular endothelium and acts as asmooth muscle relaxant. PGI₂ elicits its effects on target cells byinteraction with its G protein-coupled receptor (IP), which has atypical seven-transmembrane structure (Narumiya et al, 1999). The IPreceptor can stimulate both Gs and Gq species of G proteins causing anincrease in cAMP generation and in phosphatidylinositol response.

PGI₂ has been implicated in the aetiology of menorrhagia, which is amajor problem in women's reproductive health, accounting for 11% of allgynaecological referrals and incurring drug costs amounting to £7 m in1995 (Cooper et al, 2001). There is evidence for increased synthesis ofPGI₂ (Smith et al, 1981) or an increase in PGI₂ concentration relativeto thromboxane A₂ (Makarainen & Ylikorkala 1986) in women withmenorrhagia compared with controls. However, little information isavailable on the temporal or spatial expression of PGIS or IP in theendometrium across the menstrual cycle.

The pharmacology, molecular biology and signal transduction of theplatelet prostacyclin receptor are reviewed by Armstrong (1996,Pharmacol. Ther. 72(3), p171-191).

The cloning and sequence of the human IP receptor has been reported inEP 0 753 528 A1 (Ono Pharmaceutical Co.); U.S. Pat. Nos. 5,728,808 and6,365,360 (assigned to Merck Frosst Canada & Co.); Boie et al “Cloningand expression of a cDNA for the human prostanoid IP receptor” J. Biol.Chem. 269 (16), 12173-12178 (1994); Katsuyama et al, “Cloning andexpression of a cDNA for the human prostacyclin receptor” FEBS Lett. 344(1), 74-78 (1994);

Nakagawa et al, “Molecular cloning of human prostacyclin receptor cDNAand its gene expression in the cardiovascular system” Circulation 90(4), 1643-1647 (1994); Duncan et al, “Chromosomal localization of thehuman prostanoid receptor gene family” Genomics 25 (3), 740-742 (1995);and Ogawa et al, “Structural organization and chromosomal assignment ofthe human prostacyclin receptor gene” Genomics 27 (1), 142-148 (1995),all of which are incorporated herein by reference.

The cDNA sequence of the human IP receptor is given in Genbank AccessionNo. NM_(—)000960, and the amino acid sequence given in Genbank AccessionNo. NP_(—)000951. Some sequence variation has been identified within thehuman IP receptor gene, as is noted in Genbank Accession No.NM_(—)000960.

The cloning and sequence of human PGIS has been reported in Miyata etal, “Molecular cloning and expression of human prostacyclin synthase”Biochem. Biophys. Res. Commun. 200 (3): 1728-1734 (1994); Nakayama etal, “Organization of the human prostacyclin synthase gene” Biochem.Biophys. Res. Commun. 221 (3): 803-806 (1996); Yokoyama et al, “Humangene encoding prostacyclin synthase (PTGIS): genomic organization,chromosomal localization, and promoter activity” Genomics 36 (2):296-304 (1996); Wang et al, “Organization of the gene encoding humanprostacyclin synthase” Biochem. Biophys. Res. Commun. 226 (3): 631-637(1996); and Chevalier et al, “Characterization of new mutations in thecoding sequence and 5′-untranslated region of the human prostacylcinsynthase gene (CYP8A1)” Hum. Genet. 108 (2): 148-155 (2001).

The cDNA sequence of human PGIS receptor is given in Genbank AccessionNo. NM_(—)000961, and the amino acid sequence given in Genbank AccessionNo. NP_(—)000952. Some sequence variation has been identified within thehuman PGIS gene, as is noted in Genbank Accession No. NM_(—)000961.

We have now shown that both PGIS and IP are up-regulated in the earlyproliferative phase of the menstrual cycle, with increased signalling ofthe IP receptor via the cAMP pathway during this phase.

These observations have led the inventors to the surprising andunexpected belief that antagonising the IP receptor can combatpathological conditions of the uterus.

Antagonists of the IP receptor have been suggested for use in treatmentof a range of bladder disorders, to prevent conditions associated withexcessive bleeding such as haemophilia and haemorrhaging, to relievehypotension related to septic shock, to reduce oedema formation, and tobe useful in respiratory allergies and respiratory conditions such asasthma, inflammation, and in pain conditions such as inflammatory painand premenstrual pain (WO 02/40453; WO 02/070514; WO 02/070500; WO01/68591, EP 0 902 018; U.S. Pat. No. 6,184,242) for cardiovasculardisease (WO 79/00744) for ameliorating neuropathic disorders (WO01/10433) and treating neurodegenerative diseases (WO 01/10455).

They have not, however, been previously suggested to be useful incombating uterine pathological conditions, such as uterine cancers,endometriosis, fibroids, menorrhagia and dysmenorrhoea.

Current treatment of uterine pathologies includes the use ofCOX-inhibitors.

While COX-inhibitors have shown some therapeutic potential, they preventthe synthesis of a number of prostaglandins, of which only some areharmful, and some have beneficial effects. There is thus a need in theart for methods for treating uterine pathologies by specificallyinhibiting the action of specified prostaglandins.

A first aspect of the invention provides a method combating apathological condition of the uterus in a female individual, the methodcomprising administering to the individual at least one agent that is anantagonist of the IP receptor and/or an inhibitor of PGIS.

The IP receptor is the receptor that binds prostaglandin I₂ (PGI₂).

The pathological conditions of the uterus treatable by the methods ofthe invention include, but are not limited to, any pathologicalcondition wherein IP receptors are upregulated in proliferating tissue.

Typically, the pathological condition of the uterus is any one ofuterine cancer such as uterine carcinoma, menorrhagia, dysmenorrhoea, anendometrial pathological condition such as endometriosis includingadenomyosis, or a myometrial pathological condition such as fibroids(leiomyomas) or leiomyosarcomas which are fibroids which have becomemalignant. Thus, typically, the uterine pathological condition is onewhich is associated with abnormal growth of cells of the myometrium orendometrium.

Certain uterine pathological conditions are believed to be associatedwith overproliferation of the epithelium.

It is believed that premenstrual pain is not a pathological condition ofthe uterus.

Premenstrual pain is typically experienced in the days precedingmenstruation, which is distinguished from the uterine pathologicalcondition dysmenorrhoea that occurs during menstruation. However, forthe avoidance of doubt, the term “pathological condition of the uterus”or “uterine pathological condition” as used herein does not includepremenstrual pain.

In one particular embodiment, the invention includes a method ofcombating a pathological condition of the uterus other than menorrhagiain a female individual, the method comprising administering to theindividual at least one agent that is an antagonist of the IP receptor.

By combating a pathological condition of the uterus we includealleviating symptoms of the condition (palliative use), or treating thecondition, or preventing the condition (prophylactic use).

The invention thus includes the treatment of any of a uterine cancersuch as carcinoma, menorrhagia, dysmenorrhoea, an endometrialpathological condition such as endometriosis, and a myometrialpathological condition such as fibroids, with at least one agent that isan antagonist of the IP receptor.

The invention also includes alleviating symptoms of any of a uterinecancer such as carcinoma, menorrhagia, dysmenorrhoea, an endometrialpathological condition such as endometriosis, and a myometrialpathological condition such as fibroids, with at least one agent that isan antagonist of the IP receptor.

The invention further includes preventing, or preventing the symptomsof, any of a uterine cancer such as carcinoma, menorrhagia,dysmenorrhoea, an endometrial pathological condition such asendometriosis, and a myometrial pathological condition such as fibroids,with at least one agent that is an antagonist of the IP receptor.

The invention thus includes combating a uterine cancer, such asendometrial carcinoma, with at least one agent that is an antagonist ofthe IP receptor.

The invention also includes combating menorrhagia with at least oneagent that is an antagonist of the IP receptor.

The invention further includes combating dysmenorrhoea with at least oneagent that is an antagonist of the IP receptor.

The invention also includes combating an endometrial pathologicalcondition, such as endometriosis, with at least one agent that is anantagonist of the IP receptor.

The invention still further includes combating a myometrial pathologicalcondition, such as fibroids, with at least one agent that is anantagonist of the IP receptor.

It is possible for an individual to have more than one uterinepathological condition, and it is possible to use the methods of thepresent invention to treat the conditions together. For example, womenoften have menorrhagia and dysmenorrhoea together and the method may beused to treat both conditions in the same patient.

The patient may be any patient who is suffering from or who is at riskfrom a uterine pathological condition. Any premenopausal orperimenopausal woman is at risk of menorrhagia and/or dysmenorrhoea;however, menorrhagia is more common at the beginning and end of awoman's reproductive life so typically there is a greater risk when awoman's periods first start and in women over 40 years of age.

The patient to be treated may be any female individual who would benefitfrom such treatment. Typically and preferably the patient to be treatedis a human female. However, the methods of the invention may be used totreat female mammals, such as the females of the following species:cows, horses, pigs, sheep, cats and dogs. Thus, the methods have uses inboth human and veterinary medicine.

Typically, the agent that is an antagonist of the IP receptor is onewhich prevents or disrupts PGI₂-mediated signalling of the IP receptor,and which is suitable to be administered to a patient.

Preferably, an agent that is an antagonist of the IP receptor preventsor reduces the binding of PGI₂ to the IP receptor. Alternatively oradditionally, the antagonist may affect the interaction between PGI₂ andthe IP receptor, or the interaction between the IP receptor and theassociated Gs or Gq protein, thus inhibiting or disrupting a PGI₂-IPmediated signal transduction pathway.

IP receptor antagonists are typically molecules which bind to the IPreceptor, compete with the binding of the natural ligand PGI₂, andinhibit or disrupt the PGI₂-IP mediated signal transduction pathway.

In a preferred embodiment, preventing PGI₂ having its effect on the IPreceptor includes occupying the PGI₂ binding site on the IP receptor,such that the natural ligand (PGI₂) is prevented from binding in a modethat would result in its normal mode of signalling.

Alternatively, the receptor antagonist may be a non-competitive IPreceptor antagonist, for example a molecule which binds to the IPreceptor without preventing PGI₂ binding thereto, but which disrupts theinteraction between PGI₂ and the IP receptor, thus inhibiting ordisrupting PGI₂-IP mediated signal transduction pathway.

Further alternatively, the non-competitive IP receptor antagonist may bea molecule which binds to the IP receptor and which disrupts theinteraction between the IP receptor and the associated G protein, thusinhibiting or disrupting IP mediated signal transduction pathway.

In an alternative preferred embodiment, the agent may be an antagonistof PGI₂. PGI₂ antagonists are typically molecules which bind to PGI₂ andprevent or reduce PGI₂ binding to its receptor, which inhibits ordisrupts the PGI₂-IP mediated signal transduction pathway. This issometimes termed the ‘soluble receptor’ approach in which typically apart of the receptor binds to PGI₂.

The receptor antagonists are typically selective to the particularreceptor and preferably have an equal or higher binding affinity to theIP receptor than does PGI₂. Although antagonists with a higher affinityfor the receptor than the natural ligand are preferred, antagonists witha lower affinity may also be used, but it may be necessary to use theseat higher concentrations.

Preferably, the IP receptor antagonists bind reversibly to the IPreceptor.

Preferably, the IP receptor antagonists are selective for the IPreceptor. Thus, typically, an IP receptor antagonist binds the IPreceptor with a higher affinity than for any other prostaglandinreceptor. Preferably, an IP receptor antagonist has at least a two-foldhigher binding affinity for the IP receptor than for any otherprostaglandin receptor. More preferably, an IP receptor antagonist hasat least a three-fold, or at least a four-fold, or at least a five-fold,or at least a six-fold, or at least a seven-fold, or at least aneight-fold, or at least a nine-fold, or at least a ten-fold, or at leasta fifty-fold, or at least a 100-fold, or at least a 500-fold, or atleast a 1,000-fold higher binding affinity for the IP receptor than forany other prostaglandin receptor. Preferably, an IP receptor antagonistbinds the IP receptor but does not substantially bind any otherprostaglandin receptor.

EP 0 902 018 A2 (F. Hoffman-La Roche AG) discloses2-(Arylphenyl)amino-imidazoline derivatives which are IP receptorantagonists. All of the disclosure in EP 0 902 018 A2 relating to IPreceptor antagonists, is hereby incorporated herein by reference.

U.S. Pat. No. 6,184,242 (assigned to Syntex USA) discloses2-(substituted-phenyl)amino-imidazoline derivatives which are IPreceptor antagonists. All of the disclosure in U.S. Pat. No. 6,184,242relating to IP receptor antagonists, is hereby incorporated herein byreference.

WO 02/070514 (F. Hoffman-La Roche AG) discloses alkoxycarbonylaminoheteroaryl carboxylic acid derivatives which are IP receptorantagonists. Of these, the compounds listed in the table on page 18 arepreferred. All of the disclosure in WO 02/070514 relating to IP receptorantagonists, is hereby incorporated herein by reference.

WO 02/070500 (F. Hoffman-La Roche AG) discloses alkoxycarbonylaminobenzoic acid or alkoxycarbonylamino tetrazolyl phenyl derivatives whichare IP receptor antagonists. Of these, the compounds listed in the tableon page 24 are preferred. All of the disclosure in WO 02/070500 relatingto IP receptor antagonists, is hereby incorporated herein by reference.

WO 02/40453 (F. Hoffman-La Roche AG) discloses substituted2-phenylaminoimidazoline phenyl ketone derivatives which are IP receptorantagonists. Of these, the compounds listed in the table spanning pages26-28 are preferred. All of the disclosure in WO 02/40453 relating to IPreceptor antagonists, is hereby incorporated herein by reference.

WO 01/68591 (F. Hoffman-La Roche AG) discloses carboxylic acidderivatives which are IP receptor antagonists. Of these, the compoundslisted in the table on page 40 are preferred. All of the disclosure inWO 01/68591 relating to IP receptor antagonists, is hereby incorporatedherein by reference.

WO 01/10433 (Teijin, Inc.) discloses prostacyclin derivatives with anamino or amido group at the extremity of the α chain which may be IPreceptor antagonists. All of the disclosure in WO 01/10433 relating toIP receptor antagonists, is hereby incorporated herein by reference.

WO 01/10445 (Teijin, Inc.) discloses 15(R)-isocarbacyclin and15-deoxy-isocarbacyclin derivatives which may be IP receptorantagonists. All of the disclosure in WO 01/10445 relating to IPreceptor antagonists, is hereby incorporated herein by reference.

WO 79/00744 (Research Corporation) discloses 6,9-thiaprostacyclinanalogues and derivatives thereof which are stated as being antagonistsfor natural prostacyclin. All of the disclosure in WO 79/00744 relatingto IP receptor antagonists, is hereby incorporated herein by reference.

Corsini et al (1987 and 1998) describe (5Z)-carbacyclin as a partialagonist of the PGI₂ receptor which also displays antagonistic properties(Corsini et al (1987) Br. J. Pharmac. 90, p225-261; Corsini et al(1988), Biomedica biochimica acta 1988, 47(10-11) pS104-7).

Corsini et al (1987) also mention that the PGI₂ analogue FCE 22176((5Z)-13,14-didehydro-20-methyl-carboprostacyclin) has been shown to bea competitive antagonist of PGI₂ on guinea pig trachea and atrium(Fassina et al, (1985) Eur. J. Pharmac. 113, p459-460).

Armstrong (1996, Pharmacol. Ther. 72(3), p171-191) in a review ofplatelet prostanoid receptors, mentions that the best lead in the searchfor an IP 25 antagonist has been the synthesis of2-[3-[3-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy] acetic acid (BMY 42393)which is a partial agonist at the platelet IP receptor (Seiler et al(1994), Thromb Res. 74, p115-123).

For the avoidance of doubt, indomethacin is not an IP receptorantagonist according to the invention.

In an embodiment, the IP antagonist is an antibody.

The antibody may be monoclonal or polyclonal, but is preferablymonoclonal. Given the cDNA and amino acid sequence of the human IPreceptor (Genbank Accession Nos. NM_(—)000960 and NP_(—)000951,respectively) preparation of monoclonal antibodies, including humanisedantibodies, is well within the ability of a person of average skill inthe art.

Antibodies may be prepared by known techniques, for example thosedisclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola(CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniquesand applications”, J. Hurrell (CRC Press, 1982), both of which areincorporated herein by reference.

Human IP receptor antibodies for use in the present invention can beraised against the intact IP protein or an antigenic polypeptidefragment thereof, which may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier. Typically, the antigenic polypeptide fragment of the IPreceptor is or comprises an extracellular portion of the IP receptor.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to the IP receptor protein. Fab and F(ab′)2fragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding ofan intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus,these fragments are preferred. Alternatively, IP receptor bindingfragments can be produced through the application of recombinant DNAtechnology or through synthetic chemistry. It is further appreciatedthat other antibody-like molecules may be used in the method of theinventions including, for example, antibody derivatives which retaintheir antigen-binding sites, synthetic antibody-like molecules such assingle-chain Fv fragments (ScFv) and domain antibodies (dAbs), and othermolecules with antibody-like antigen binding motifs.

The antibodies may be prepared by any of a variety of methods. Forexample, cells expressing the IP receptor or an antigenic fragmentthereof can be administered to an animal in order to induce theproduction of sera containing polyclonal antibodies. In a preferredmethod, a preparation of IP receptor protein is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity.

In the most preferred method, the antibodies are monoclonal antibodies(or IP receptor binding fragments thereof). Such monoclonal antibodiescan be prepared using hybridoma technology (Kohler et al, Nature 256:495(1975); Kohler et al, Eur. J. Immunol 6:511 (1976); Kohler et al, Eur.J. Immunol. 6:292 (1976); Hammerling et al, in: Monoclonal Antibodiesand T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681). In general,such procedures involve immunising an animal (preferably a mouse) with aIP receptor antigen or, more preferably, with an IP receptor expressingcell. It is further preferred if the mouse is a transgenic mouse whichis transgenic for the human Ig region, thus producing humanisedantibodies.

It is preferred if the anti-IP antibodies are humanised antibodies,which are suitable for administration to humans without engendering animmune response by the human against the administered immunoglobulin(Ig). Humanised forms of antibodies may include chimaeric Igs, Ig chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) that are principallycomprised of the sequence of a human Ig, and contain minimal sequencederived from a non-human Ig.

Methods for producing chimaeric antibodies are known in the art. See,for a review, Morrison, Science 229:1202 (1985); Oi et al, BioTechniques4:214 (1986); Cabilly et al, U.S. Pat. No. 4,816,567; Taniguchi et al,EP 171496;

Morrison et al, EP 173494; Neuberger et al, WO 8601533; Robinson et al,WO 870267 1; Boulianne et al, Nature 312:643 (1984); Neuberger et al,Nature 314:268 (1985).

Humanisation can be performed following the method of Winter andco-workers (Jones et al, Nature, 321:522-525 (1986); Riechmann et al,Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536(1988)), by importing rodent CDRs or CDR sequences into a humanantibody. (See also U.S. Pat. No. 5,225,539). In some instances, Fvframework residues of the human Ig are replaced by correspondingnon-human residues. Humanised antibodies can also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanised antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human Ig and all or substantially all of the framework regionsare those of a human Ig consensus sequence. The humanised antibodyoptimally also will comprise at least a portion of an Ig constant region(Fc), typically that of a human Ig (Jones et al, 1986; Riechmann et al,1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Humanised antibodies also include antibody molecules in whichessentially the entire sequences of both the light chain and the heavychain, including the CDRs, arise from human genes. Humanised monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce humanised monoclonal antibodies(see Cole, et al, 1985 In: Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96).

In addition, humanised antibodies can also be produced using techniquessuch as phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991)).

Similarly, humanised antibodies can be made by introducing human Ig lociinto transgenic animals, e.g., mice in which the endogenous Ig geneshave been partially or completely inactivated. Upon challenge, antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inMarks et al (Bio/Technology 10, 779-783 (1992)); Lonberg et al (Nature368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild etal, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (NatureBiotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.Immunol. 13 65-93 (1995)).

The preferred embodiment of such a nonhuman animal is a mouse, and istermed the Xenomouse™ as disclosed in PCT publications WO 96/33735 andWO

This animal produces B cells that secrete fully human Igs. Theantibodies can be obtained directly from the animal after immunisationwith an immunogen of interest, as, for example, a preparation of apolyclonal antibody, or alternatively from immortalised B cells derivedfrom the animal, such as hybridomas producing monoclonal antibodies.Additionally, the genes encoding the Igs with human variable regions canbe recovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogues of antibodies such as, for example,single chain Fv molecules.

A further method for producing a humanised antibody is disclosed in U.S.Pat. No. 5,916,771. It includes introducing an expression vector thatcontains a nucleotide sequence encoding a heavy chain into one mammalianhost cell in culture, introducing an expression vector containing anucleotide sequence encoding a light chain into another mammalian hostcell, and fusing the two cells to form a hybrid cell. The hybrid cellexpresses an antibody containing the heavy chain and the light chain.

Additional IP receptor antagonists may be identified using the screeningmethods described in EP 0 753 528 A1 (Ono Pharmaceutical Co.) and U.S.Pat. Nos. 5,728,808 and 6,365,360 (assigned to Merck Frosst Canada &Co.), all of which are incorporated herein by reference.

For the avoidance of doubt, by “antagonist of the IP receptor” weinclude an antagonist of PGI₂, which may be any PGI₂ antagonist that issuitable to be administered to the patient. The PGI₂ antagonists arepreferably selective to PGI₂ and typically have a higher bindingaffinity for PGI₂ than for other molecules. Although antagonists with ahigher affinity for PGI₂ than other molecules are preferred, antagonistswith a lower affinity may also be used, but it may be necessary to usethese at higher concentrations. Preferably, the PGI₂ antagonists bindreversibly to PGI₂.

Suitable PGI₂ antagonists may include extracellular fragments of the IPreceptor.

As used herein, the term ‘antagonist’ covers all types of antagonism.(G-protein-coupled receptors (GPCRs) such as prostaglandin receptors areknown to show inverse agonism which has the outcome of blocking adesired response. Thus a suitable IP antagonist for use in the presentinvention may be identified by measuring the binding of a radio-labelledIP agonist to PGI₂ with or without the purported antagonist. Secondly,IP antagonists may be identified in a functional assay eg by showingthat the effect of an IP agonist on Ca²⁺ levels is modified in thepresence of the antagonist. Thirdly IP antagonists may be identified byinhibition of epithelial cell growth in cell culture.

In an embodiment, the inhibitor of PGIS is an anti-PGIS antibody.

A suitable antibody may be the anti-PGIS antibody available as CatalogueNo. CAY-160630 from Alexis Corporation (Nottingham, UK). This is amonoclonal antibody against bovine PGIS, which has 88% sequence homologywith human PGIS.

Preferably, the anti-PGIS antibody is a humanised antibody as describedabove. Given the cDNA and protein sequence of human PGIS (GenbankAccession Nos. NM_(—)000961 and NP_(—)000952, respectively) preparationof monoclonal antibodies, including humanised antibodies, is well withinthe ability of a person of average skill in the art.

The compound U-51605 has been reported as being a PGIS inhibitor (Bayorhet al, ASGSB 2001 Annual Meeting Abstract No. 82).

Peryoxynitrite (PON) and the PON generator 3-morpholinosydnonimineN-ethylcarbamide (SIN-1) have been reported to inhibit PGIS by tyrosinenitration at the active site (Zou et al, 1998, Biochem. J. 336:507-512).

Trans-2-phenylcyclopropylamine HCl has been reported as being a PGISinhibitor (Gryglewski et al, Prostaglandins 1976, 12: 685; Hoyns & vanAlphen, Doc. Ophthol. (Netherlands) 1981, 51: 225).

All of the patent and non-patent documents referred to herein thatdescribe antagonists and inhibitors of the IP receptor or PGIS areincorporated herein, in their entirety, by reference.

EP Receptor Antagonists

PGE₂ mediates its effect on target cells through interaction withdifferent isoforms of seven transmembrane G protein coupled receptorswhich belong to the rhodopsin family of serpentine receptors. Four mainPGE₂ receptor subtypes have been identified (EP1, EP2, EP3 and EP4)which utilise alternate and in some cases opposing intracellularsignalling pathways (Coleman et al, 1994). This diversity of receptorswith opposing action may confer a homeostatic control on the action ofPGE₂ that is released in high concentrations close to its site ofsynthesis (Ashby, 1998). To-date, the role of the different PGE₂receptors, their divergent intracellular signalling pathways, as well astheir respective target genes involved in mediating the effects of PGE₂on normal or neoplastically transformed endometrial epithelial cellsremain to be elucidated fully.

We have previously studied the expression of PGE synthase and of the EP2and EP4 receptors across the menstrual cycle and have found that PGESexpression is reduced during the late secretory phase and that EP4expression is significantly higher in the late proliferative stage(Milne et al, (2001) J. Clin. Endocrinol. 86(9): 4453-4459). We havealso previously found that PGES expression and PGE₂ synthesis are alsoup-regulated in pathological conditions of the uterus in humans. Forexample, in adenocarcinoma, expression of these factors was localised tothe neoplastic epithelial cells of the uterine carcinoma tissues as wellas the endothelial cells of the microvasculature. This is associatedwith an overexpression and signalling of the EP2 and EP4 receptors inthe carcinoma tissue. We have previously suggested the use of aninhibitor of PGES or an EP2 or EP4 receptor antagonist in the treatmentor prevention of a pathological condition of the uterus (PCT/GB02/004845and PCT/GB02/004549).

In a further embodiment of the present invention, in addition to the atleast one agent that is an antagonist of the IP receptor, the individualis also administered an inhibitor of PGES and/or an antagonist of EP2 orof EP4 (which term, for the avoidance of doubt, includes an antagonistor an EP2 receptor and an antagonist of an EP4 receptor, respectively).

In one embodiment of the invention, the individual is administered aninhibitor of PGES. It has been reported by Thoren & Jakobsson (2000)Eur. J Biochem. 267, 6428-6434 (incorporated herein by reference) thatNS-398, sulindac sulphide and leukotriene C₄ inhibit PGES activity withIC₅₀ values of 20 μM, 80 μM and 5μM, respectively.

In a still further embodiment of the invention, the individual isadministered an antagonist of an EP2 receptor or an antagonist of an EP4receptor.

The prostaglandin EP2 receptor antagonist may be any suitable EP2receptor antagonist. Similarly, the prostaglandin EP4 receptorantagonist may be any suitable EP4 receptor antagonist. By “suitable” wemean that the antagonist is one which may be administered to a patient.The receptor antagonists are molecules which bind to their respectivereceptors, compete with the natural ligand (PGE₂) and inhibit theinitiation of the specific receptor-mediated signal transductionpathways. The receptor antagonists are typically selective to theparticular receptor and typically have a higher binding affinity to thereceptor than the natural ligand. Although antagonists with a higheraffinity for the receptor than the natural ligand are preferred,antagonists with a lower affinity may also be used, but it may benecessary to use these at higher concentrations. Preferably, theantagonists bind reversibly to their cognate receptor. Typically,antagonists are selective for a particular receptor and do not affectthe other receptor; thus, typically, an EP2 receptor antagonist bindsthe EP2 receptor but does not substantially bind the EP4 receptor,whereas an EP4 receptor antagonist binds the EP4 receptor but does notsubstantially bind the EP2 receptor. Preferably, the EP2 or EP4 receptorantagonist is selective for the particular receptor subtype. By this ismeant that the antagonist has a binding affinity for the particularreceptor subtype which is at least ten-fold higher than for at least oneof the other EP receptor subtypes. Thus, selective EP4 receptorantagonists have at least a ten-fold higher affinity for the EP4receptor than any of the EP1, EP2 or EP3 receptor subtypes.

It is particularly preferred that the EP2 or EP4 receptor antagonist isselective for its cognate receptor.

EP2 receptor antagonists include AH6809 (Pelletier et al (2001) Br. J.Phamacol. 132, 999-1008).

EP4 receptor antagonists include AH23848B (developed by Glaxo) andAH22921X (Pelletier et al (2001) Br. J. Pharmacol. 132, 999-1008. Thechemical name for AH23848B is ([1alpha(z),2beta5alpha]-(±)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxo-cyclopentyl]-4-heptenoics acid) (see Hillock & Crankshaw (1999) Eur. J. Pharmacol. 28, 99-108).EP4RA (Li (2000) Endocrinology 141, 2054-61) is an EP(4) -selectiveligand (Machwate et al (2001) Mol. Pharmacol. 60: 36-41). Theomega-substituted prostaglandin E derivatives described in WO 00/15608(EP 1 114 816) (Ono Pharm Co Ltd) bind EP4 receptors selectively and maybe EP4 receptor antagonists.

Peptides described in WO 01/42281 (Hopital Sainte-Justine) eg: IFTSYLECL(SEQ ID No 1), IFASYECL (SEQ ID No 2), IFTSAECL (SEQ ID No 3), IFTSYEAL(SEQ ID No 4), ILASYECL (SEQ ID No 5), IFTSTDCL (SEQ ID No 6), TSYEAL(SEQ ID No 7) (with 4-biphenyl alanine), TSYEAL (SEQ ID No 7) (withhomophenyl alanine) are also described as EP4 receptor antagonists, asare some of the compounds described in WO 00/18744 (Fujisawa Pharm CoLtd). The 5-thia-prostaglandin E derivatives described in WO 00/03980(EP 1 097 922) (Ono Pharm Co Ltd) may be EP4 receptor antagonists.

EP4 receptor antagonists are also described in WO 01/10426 (Glaxo), WO00/21532 (Merck) and GB 2 330 307 (Glaxo).

WO 00/21532 describes the following as EP4 receptor antagonists:

5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt;

5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one;

5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.

GB 2 330 307 describes [1α(Z),2β,5α]-(±)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoicacid and[1R[1α(z),2β,5α]]-(−)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoicacid.

WO 00/18405 (Pharmagene) describes the EP4 receptor antagonists AH22921and AH23848 (which are also described in GB 2 028 805 and U.S. Pat. No.4,342,756). WO 01/72302 (Pharmagene) describes further EP4 receptorantagonists, for example those described by reference to, and includedin the general formula (I) shown on page 8 et seq.

In an embodiment, when an inhibitor of PGES and/or an antagonist of EP2or of EP4 is administered to a patient in addition to the at least oneagent that is an antagonist of the IP receptor, the dose of eachcompound may be the same as would be administered individually withoutreference to the other compound. Alternatively and preferably, lowerdoses may be administered.

All of the patents and other documents referred to herein that describeantagonists or inhibitors of EP2 or EP4 and PGES, are incorporatedherein, in their entirety, by reference.

FP Receptor Antagonists

The FP prostaglandin receptor has been studied in a variety of tissuesincluding the bovine corpus luteum (Sharif et al 1998, J. Pharmacol.Exp. Ther. 286: 1094-1102); human uterus (Senior et al 1992, Br. J.Pharmacol. 108: 501-506); rabbit jugular vein (Chen et al 1995, Br. J.Pharmacol. 116: 3035-3041); various human ocular tissues (Davis & Sharif1999, J. Ocular Pharmacol. Ther. 15: 323-336); and in mouse Swiss 3T3fibroblasts (Griffin et al 1997, J. Pharmacol. Exp. Ther. 281: 845-854);and in rat vascular smooth muscle cells (A7r5) (Griffin et al 1998, J.Pharmacol. Exp. Ther. 286: 411-418).

Potent, selective synthetic agonists at some prostaglandin receptorshave been characterised in both in vitro and in vivo models (Coleman etal 1994, Pharmacol. Rev. 46: 205-229). For instance fluprostenol or itsenantiomer (eg AL-5848) (Sharif et al 1999, J Pharm. Pharmacol., 51:685-694) and cloprostenol (Coleman et al 1994; Sharif et al 1998) arepotent and selective FP receptor agonists. Since most naturalprostaglandins show rather limited selectivity for their preferredreceptor among this receptor family, the few reported selectiveprostaglandin receptor agonists have been very valuable tools fordiscriminating discrete functional responses coupled to their respectivereceptors. However, conclusive identification of the particularreceptors mediating prostaglandin-stimulated functional responsesrequires potent and selective antagonists (Kenakin 1996, Pharmacol. Rev.48: 413:463).

The recent identification and commercial development of selective FPreceptor agonists as potent and highly efficacious drugs for thetreatment of elevated intraocular pressure (Bito 1997, Surv. Ophthalnol.41 (Suppl. 22): S1-S14; Hellberg et al 1998, Invest. Ophthalmol. Vis.Sci. 39 (Suppl.): 1961) has considerably advanced our knowledge of FPreceptor-coupled pharmacological actions. However, the function of theFP receptor is not fully understood, due in part to significant speciesdifferences in the tissue distribution of this receptor (Ocklind et al1996, Invest. Ophthalmol. Vis. Sci. 37: 716-726; Davis & Sharif 1999;Sharif et al 1999).

Griffin et al (J. Pharmacol. Exp. Ther. 1999, 290: 1278-1284) reportedthe discovery of a selective FP receptor antagonist (AL-8810) ofmicromolar potency. Sharif et al (J. Pharm. Pharmacol. 2000, 52:1529-1539) describe another analogue of PGF_(2α) (AL-3138; Ro-22-6641;11-deoxy-16-fluoro PGF_(2α)) which is a partial agonist of low efficacyand which also functions as an FP receptor antagonist. AL-3138 being arelatively selective agent may be a valuable FP receptor antagonist toolfor investigating the specific function of the FP receptor in variousbiological systems.

We have also previously shown that expression of the PGF_(2α) receptorin the uterus across the menstrual cycle demonstrates higher level ofthe receptor during the proliferative phase of the endometrium comparedwith other stages. Expression in uterine carcinoma tissue issignificantly elevated compared with normal uterine tissue. Using anendometrial epithelial cell line, we have demonstrated that PGF_(2α)induces proliferation of epithelial cells. This proliferation can beinhibited by using specific inhibitors of the PLC signalling pathway.These observations demonstrate the possibility of antagonising thePGF_(2α) (FP) receptor to combat pathological conditions of the uterus,such as to reduce the proliferation of epithelial cells in uterinecarcinoma, as we have previously suggested (GB 0208785.6). Antagonistsof the FP receptor have also been suggested for treating or preventingpremature delivery of a foetus and dysmenorrhoea, acting by themechanism of relaxation of smooth muscle (WO 99/32640 and WO 00/17348).We have also suggested using antagonists of the FP receptor to treatmenorrhagia (GB 0208783.1).

In a further embodiment of the present invention, in addition to the atleast one agent that is an antagonist of the IP receptor, and optionallyan inhibitor of

PGES and/or an antagonist of EP2 or of EP4, the individual is alsoadministered at least one agent that is an antagonist of the FPreceptor.

Typically, the agent is one which prevents or disrupts PGF_(2α)-mediatedsignalling of the FP receptor. Preferably, an agent that is anantagonist of the FP receptor prevents or reduces the binding ofPGF_(2α) to the FP receptor. Alternatively or additionally, the agentmay affect the interaction between PGF_(2α) and the FP receptor, or theinteraction between the FP receptor and the associated G_(αq) protein,thus inhibiting or disrupting the PGF_(2α)-FP mediated signaltransduction pathway.

In one preferred embodiment, the agent that is an antagonist of the FPreceptor may be an antagonist of the FP receptor. FP receptorantagonists are typically molecules which bind to the FP receptor,compete with the binding of the natural ligand PGF_(2α), and inhibit ordisrupt the PGF_(2α)-FP mediated signal transduction pathway.

In one preferred embodiment, antagonising the FP receptor includesoccupying the PGF_(2α) binding site on the prostaglandin receptor, suchthat the natural ligand (PGF_(2α)) is prevented from binding in a modethat would result in its normal mode of signalling via Gq/Gq_(II)through inosityl phosphate and subsequent mobilisation of intracellularcalcium.

Alternatively, the receptor antagonist may be a molecule which binds tothe FP receptor without preventing PGF_(2α) binding thereto, but whichdisrupts the interaction between PGF_(2α) and the FP receptor, thusinhibiting or disrupting PGF_(2α)-FP mediated signal transductionpathway.

Further alternatively, the FP receptor antagonist may be a moleculewhich binds to the FP receptor and which disrupts the interactionbetween the FP receptor and the associated G_(αaq) protein, thusinhibiting or disrupting FP mediated signal transduction pathway.

For the avoidance of doubt, by “antagonist of the FP receptor” weinclude an antagonist of PGF_(2α). PGF_(2α) antagonists are typicallymolecules which bind to PGF_(2α) and prevent or reduce PGF_(2α) bindingto its receptor, which inhibits or disrupts the PGF_(2α)-FP mediatedsignal transduction pathway. This is often termed the ‘soluble receptor’approach in which typically either a part of the receptor or an antibodybinds to PGF_(2α).

Alternatively, the PGF_(2α) antagonist may be a molecule which binds toPGF_(2α) without preventing or reducing the binding of PGF_(2α) to theFP receptor, but which disrupts the interaction between PGF_(2α) and theFP receptor such that the PGF_(2α)-FP mediated signal transductionpathway is inhibited or disrupted. This could be a molecule which bindsin a covalent fashion to PGF_(2α) and has no effect on binding potencybut effects the G-protein/IP/Ca²⁺ mechanisms.

The receptor antagonists are typically selective to the particularreceptor and preferably have an equal or higher binding affinity to theFP receptor than does PGF_(2α). Although antagonists with a higheraffinity for the receptor than the natural ligand are preferred,antagonists with a lower affinity may also be used, but it may benecessary to use these at higher concentrations. Preferably, theantagonists bind reversibly to the FP receptor. Preferably, antagonistsare selective for a particular receptor and do not affect otherreceptors; thus, typically, an FP receptor antagonist binds the FPreceptor but does not substantially bind any other receptor.

The peptides listed in Table 1 are reported to be antagonists of the FPreceptor that disrupt the interaction between the FP receptor and theassociated G_(αq) protein (WO 99/32640 and WO 00/17438). The amino acidsare indicated according to the standard IUPAC single letter convention,and X is cyclohexyl alanine. Lower case letters indicate L-amino acidsand capital letters indicate D-amino acids. (For the avoidance of doubt,it is only for the peptides in Table 1 where this nomenclature is used.All other peptides, unless indicated to the contrary, are comprised ofL-amino acids.) All of the disclosure in WO 99/32640 and WO 00/17438relating to specific peptides as FP receptor antagonists, is herebyincorporated herein by reference. TABLE 1 PCP-1 rvkfksqqhrqgrshhlem (SEQID No 8) PCP-2 rkavlknlyklasqccgvhvislhiw (SEQ ID No 9)elssiknslkvaaisespvaeksast PCP-3 clseeakearrindeierqlrrdkrd (SEQ ID No10) arre-NH₂ PCP-4 kdtilqlnlkeynlv-NH₂ (SEQ ID No 11) PCP-8 ilghrdyk(SEQ ID No 12) PCP-10 wedrfyll (SEQ ID No 13) PCP-13 ILGHRDYK (SEQ ID No14) PCP-14 YQDRFYLL (SEQ ID No 15) PCP-13.7 ILAHRDYK (SEQ ID No 16)PCP-13.8 ILaHRDYK (SEQ ID No 17) PCP-13.11 ILGFRDYK (SEQ ID No 18)PCP-13.13 ILGHKDYK (SEQ ID No 19) PCP-13.14 ILGHRNYK (SEQ ID No 20)PCP-13.18 ILGHQDYK (SEQ ID No 21) PCP-13.20 ILGHRDY-amide (SEQ ID No 22)PCP-13.21 ILGHRDYK-amide (SEQ ID No 23) PCP-13.22 ILGWRDYK (SEQ ID No24) PCP-13.24 ILGXRDYK (SEQ ID No 25) PCP-15 SNVLCSIF (SEQ ID No 26)

When the antagonist comprises a peptide, such as those mentioned inTable 1, the antagonist may also comprise protein fusions orpeptidomimetics thereof.

PGF_(2α) dimethyl amide, obtained from Cayman Chemical, Ann Arbor,Mich., USA was reported to be a PGF_(2α) receptor antagonist (Arnould etal, (2001) Am. J. Pathol., 159(1): 345-357).

AL-8810 ((5Z, 13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16, 17, 18, 19,20-pentanor-5,13-prostadienoic acid) obtained from Alcon Research wasreported to be a weak partial agonist of the PGF_(2α) receptor and ahighly selective antagonist of the PGF_(2α) receptor. AL-8810 wasreported not to significantly inhibit functional responses ofprostaglandin receptors TP, DP, EP2 or EP4 at high 10 μM concentration(Griffin et al, (1999) J. Pharmacol. Exp. Ther., 260(3): 1278-1284).

AL-3138 (1 1-deoxy-16-fluoro PGF_(2α)) was reported to be a weak partialagonist of the PGF_(2α) receptor, and also a highly selective antagonistof the PGF_(2α) receptor (Sharifet al, (2000) J. Pharm. Pharmacol.,52(12): 1529-1539).

Phloretin was reported to be a PGF_(2α) receptor antagonist (Kitanaka etal (1993) J. Neurochem. 60(2): 704-708).

The sulfonylurea glibenclamide was reported to be a PGF_(2α) receptorantagonist (Delaey and Van de Voorde (1995), Eur. J. Pharmacol. 280(2):179-184). The sulfonylureas tolbutamide and tolazamide were reported tobe very weak antagonists of the FP receptor. (Sharif et al (2000) J.Pharm. Pharmacol., 52(12): 1529-1539).

PGF_(2α) dimethyl amine was reported to be a PGF_(2α) receptorantagonist (Stinger et al (1992), J. Pharmacol. Exp. Ther., 220:521-525).

(E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]methylen]amino]oxy]pentanoic acid, also known as ridogrel,obtained from Jannsen Pharmaceutica, was reported to be a PGF_(2α)receptor antagonist (Jannsens et al, (1990), Thrombosis and Haemostasis,64(1): 91-96).

The compound PHG113 was reported to be a selective PGF_(2α) receptorantagonist (Quiniou et al, (2001) Pediatric Research, 49(2): 452A.

EP-128479 describes pyrazolyl-methyl-ergoline derivatives which arereported to be PGF_(2α) receptor antagonists. All the disclosure inEP-128479 relating to pyrazolyl-methyl-ergoline derivatives as PGF_(2α)receptor inhibitors, is hereby incorporated herein by reference.

The PGF_(2α) antagonists (which as noted above are included in the termFP receptor antagonist) are preferably selective to PGF_(2α) andtypically have a higher binding affinity for PGF_(2α) than for othermolecules. Although antagonists with a higher affinity for PGF_(2α) thanother molecules are preferred, antagonists with a lower affinity mayalso be used, but it may be necessary to use these at higherconcentrations. Preferably, the PGF_(2α) antagonists bind reversibly toPGF_(2α).

PGF_(2α) antagonists include anti-PGF_(2α) antibodies such as rabbitpolyclonal anti-PGF_(2α) antibodies from Oxford Biomedical Research,Inc., Oxford, UK (Arnould et al, Am. J. Pathol. 2001 159(1): 345-357).Arnould et al state that, according to the manufacturer, the specificityof the antibody is very high and the cross-reactivity with otherprostanoid derivatives is <1%.

JP 04077480; JP 08176134; JP 01199958; JP 01050818; and JP 63083081 eachdescribe phthalide derivatives that are reported to be PGF_(2α)inhibitors. All the disclosure in JP 04077480; JP 08176134; JP 01199958;JP 01050818; and JP 63083081 relating to phthalide derivatives asPGF_(2α) inhibitors, is hereby incorporated herein by reference.

WO 91/13875 describes (iso) quinoline sulphonamide compounds which arereported to be PGF_(2α) inhibitors. All the disclosure in WO 91/13875relating to (iso) quinoline sulphonamide compounds as PGF_(2α)inhibitors, is hereby incorporated herein by reference.

Some of the compounds reported as being inhibitors or antagonists ofPGF_(2α) may, in fact, be antagonists of the PGF_(2α) (FP) receptor, asused and defined herein. References to such compounds as inhibitors orantagonists of PGF_(2α) should therefore be considered as references toFP receptor antagonists.

All of the patents and other documents referred to herein that describeFP antagonists or inhibitors, are incorporated herein, in theirentirety, by reference.

COX-2 Inhibitors

We have previously shown that cyclooxygenase-2 (COX-2) synthesis isup-regulated in pathological conditions of the uterus in humans. Forexample, in adenocarcinoma of the human uterus, expression was localisedto the neoplastic epithelial cells of the uterine carcinoma tissues aswell as the endothelial cells of the microvasculature. We havepreviously suggested the use of an inhibitor of a COX-2 inhibitor,typically in conjunction with a PGES or an EP2 or EP4 receptorantagonist, in the treatment or prevention of a pathological conditionof the uterus (PCT/GB02/004549).

In a further embodiment of the present invention, in addition to the atleast one agent that is an antagonist of the IP receptor, and optionallyan inhibitor of PGES and/or an antagonist of EP2 or of EP4, and alsooptionally an agent that is an antagonist of the FP receptor, theindividual is also administered a COX-2 inhibitor.

Preferably the inhibitor is selective for COX-2.

The compound may selectively inhibit COX-2 function at any level.Suitably, the compound selectively inhibits COX-2 enzyme activity.

By “selectively inhibits COX-2 enzyme activity” we mean that thecompound preferably inhibits COX-2 in preference to other cyclooxygenaseenzymes, in particular in preference to COX-1. The COX-1 gene and thesequence of its polypeptide product are described in Yokoyama and Tanabe(1989) Biochem. Biophys. Res. Comm. 165, 888-894 incorporated herein byreference. COX-1 is also called PGHS-1. The COX-2 gene and the sequenceof its polypeptide product are described in O'Banion et al (1991) J.Biol. Chem. 266, 23261-23267 incorporated herein by reference. COX-2 isalso called PGHS-2.

Conveniently, the compound which selectively inhibits COX-2 enzymeactivity is at least ten times better at inhibiting COX-2 than COX-1;preferably it is at least fifty times better; preferably it is at leastone hundred times better; still more preferably it is at least onethousand times better and in greater preference it is at least tenthousand times better.

It is most preferred if the COX-2 inhibitor compound has substantiallyno inhibitory activity against the COX-1 enzyme.

Conveniently, the compound selectively inhibits COX-2 enzyme production.The compound may, for example, selectively prevent transcription of theCOX-2 or it may selectively prevent translation of the COX-2 message.

By “selectively inhibits COX-2 enzyme production” we mean that thecompound preferably inhibits the production of COX-2 in preference toother cyclo-oxygenases, in particular in preference to the production ofCOX-1.

Conveniently, the compound which selectively inhibits COX-2 enzymeproduction is at least ten times better at inhibiting COX-2 productionthan COX-1 production; preferably it is at least fifty times better;more preferably it is at least one hundred times better; more preferablystill it is at least one thousand times better; and in greaterpreference it is at least ten thousand times better.

It is most preferred if the compound has substantially no inhibitoryactivity against COX-1 enzyme production.

A particularly preferred embodiment is wherein the COX-2 inhibitorcompound is any one of nimesulide, 4-hydroxynimesulide, flosulide, andmeloxicam.

Nimesulide is N-(4-nitro-2-phenoxyphenyl) methanesulfonamide (alsocalled 4-nitro-2-phenoxymethanesulfonanilide). Nimesulide is 100-foldmore specific for COX-2 inhibition than for COX-1 inhibition. Nimesulideis manufactured by Boehringer.

Flosulide is 6-(2,4-difluorophenoxy)-5-methyl sulphonylamino-1-indanone(also known as N-6-(2,4-difluorophenoxy)-1-oxo-indan-5-ylmethane-sulphonamide). Flosulide is 1000-fold more specific for COX-2inhibition than for COX-1 inhibition. Flosulide is manufactured by CibaGeigy.

Meloxicam is4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide1,1-dioxide. Meloxicam is 1000-fold more specific for COX-2 inhibitionthan for COX-1 inhibition. Meloxicam is manufactured by Boehringer.

The synthesis of nimesulide is well known and is described in U.S. Pat.No. 3,840,597; the synthesis of flosulide is well known and is describedin GB 2 092 144; and the synthesis of meloxicam is well known and isdescribed in U.S. Pat. No. 4,233,299.

Other COX-2-specific inhibitors which may be useful in the practice ofthe invention include:

L 475 L337 which is 500-fold more specific for COX-2 inhibition than forCOX-1 inhibition. This is manufactured by Merck Frost.

Vioxx, sold by Merck, is also a suitable COX-2 inhibitor.

SC 58125 Celecoxib which is 100-fold more specific for COX-2 inhibitionthan for COX-1 inhibition. Celecoxib is manufactured by Searle.

NS 398 which is manufactured by Taisho and which is very highlyselective for COX-2.

DuP 697, which is COX-2-selective and is manufactured by DuPont.

Nimesulide, flosulide and meloxicam are COX-2 enzyme inhibitors,probably competitive inhibitors.

All of the patents and other documents referred to herein that describeCOX-2 inhibitors, are incorporated herein, in their entirety, byreference.

It is appreciated that one or two or more agents that are antagonists ofthe IP receptor and/or PGIS inhibitors may be administered to thepatient.

Typically, the agent(s) that is an antagonist of the IP receptor and/orPGIS inhibitor(s) is (are) administered in a quantity and frequency suchthat an effective dose is delivered to at least 90% of the IP receptors(ED₉₀). The potency of the molecule would dictate the dose, as would theformulation and route of administration.

Optionally, other treatment agents may also be administered to thepatient, as described above. These may also be considered treatmentagents of the invention. It will also be appreciated that when more thanone treatment agent is administered to the patient, they may beadministered sequentially or in combination.

The treatment agent(s) are administered in an effective amount to combatthe undesired pathological condition of the uterus. Thus, the treatmentagents may be used to alleviate symptoms (ie are used palliatively), ormay be used to treat the condition, or may be used prophylactically toprevent the condition. The treatment agent may be administered by anysuitable route, and in any suitable form.

The aforementioned treatment agents for use in the invention or aformulation thereof may be administered by any conventional methodincluding oral and parenteral (eg subcutaneous or intramuscular)injection. The treatment may consist of a single dose or a plurality ofdoses over a period of time. The dose to be administered is determinedupon consideration of age, body weight, mode of administration, durationof the treatment and pharmacokinetic and toxicological properties of thetreatment agent or agents. The treatment agents are administered at adose (or in multiple doses) which produces a beneficial therapeuticeffect in the patient. Typically, the treatment agents are administeredat a dose the same as or similar to that used when the treatment agentis used for another medical indication. In any event, the dose suitablefor treatment of a patient may be determined by the physician.

Whilst it is possible for a treatment agent of the invention to beadministered alone or in combination with other treatment agents, it ispreferable to present it or them as a pharmaceutical formulation,together with one or more acceptable carriers. The carrier(s) must be“acceptable” in the sense of being compatible with the treatment agentof the invention and not deleterious to the recipients thereofTypically, the carriers will be water or saline which will be sterileand pyrogen free.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the treatmentagent or agents with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient (ie treatmentagent or agents) with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (eg sodium starch glycolate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide desiredrelease profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier. Buccal administration is also preferred.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

Certain of the treatment agents are proteins or peptides. Proteins andpeptides may be delivered using an injectable sustained-release drugdelivery system. These are designed specifically to reduce the frequencyof injections. An example of such a system is Nutropin Depot whichencapsulates recombinant human growth hormone (rhGH) in biodegradablemicrospheres that, once injected, release rhGH slowly over a sustainedperiod.

The protein and peptide can be administered by a surgically implanteddevice that releases the drug directly to the required site. Forexample, Vitrasert releases ganciclovir directly into the eye to treatCMV retinitis. The direct application of this toxic agent to the site ofdisease achieves effective therapy without the drug's significantsystemic side-effects.

Electroporation therapy systems can also be employed for theadministration of proteins and peptides. A device which delivers apulsed electric field to cells increases the permeability of the cellmembranes to the drug, resulting in a significant enhancement ofintracellular drug delivery.

Proteins and peptides can be delivered by electroincorporation whichoccurs when small particles of up to 30 microns in diameter on thesurface of the skin experience electrical pulses identical or similar tothose used in electroporation. In electroincorporation, these particlesare driven through the stratum corneum and into deeper layers of theskin. The particles can be loaded or coated with drugs or genes or cansimply act as “bullets” that generate pores in the skin through whichthe drugs can enter.

An alternative method of protein and peptide delivery is the ReGelinjectable system that is thermo-sensitive. Below body temperature,ReGel is an injectable liquid while at body temperature it immediatelyforms a gel reservoir that slowly erodes and dissolves into known, safe,biodegradable polymers. The treatment agent is delivered over time asthe biopolymers dissolve.

Protein and peptide pharmaceuticals can also be delivered orally. Theprocess employs a natural process for oral uptake of vitamin B₁₂ in thebody to co-deliver proteins and peptides. By riding the vitamin B₁₂uptake system, the protein or peptide can move through the intestinalwall. Complexes are synthesised between vitamin B₁₂ analogues and thedrug that retain both significant affinity for intrinsic factor (IF) inthe vitamin B₁₂ portion of the complex and significant bioactivity ofthe drug portion of the complex.

Proteins and polypeptides can be introduced to cells by “Trojanpeptides”.

These are a class of polypeptides called penetratins which havetranslocating properties and are capable of carrying hydrophiliccompounds across the plasma membrane. This system allows directtargeting of oligopeptides to the cytoplasm and nucleus, and may benon-cell type specific and highly efficient. See Derossi et al (1998),Trends Cell Biol 8, 84-87.

The treatment agents or formulations may also be administeredtransdermally, eg as a patch, gel, lotion, cream or oil.

It is preferred if the treatment agent (or agents) is administeredorally.

It is further preferred if the treatment agent (or agents) orformulation thereof is administered to the female reproductive system.For example, the treatment agent(s) may suitably be administeredintravaginally using, for example, a gel or cream or paste or foam orspray or pessary or vaginal ring or tampon. The treatment agent may alsoadvantageously be administered by intrauterine delivery, for exampleusing methods well known in the art such as an intrauterine device.

Typically, the gel or cream is one which is formulated foradministration to the vagina. It may be oil based or water based.Typically, the treatment agent(s) is present in the cream or gel in asufficient concentration so that an effective amount is administered ina single (or in repeated) application.

Typically, the vaginal ring comprises a polymer which formed into a“doughnut” shape which fits within the vagina. The treatment agent (oragents) is present within the polymer, typically as a core, which maydissipate through the polymer and into the vagina and/or cervix in acontrolled fashion. Vaginal rings are known in the art.

Typically, the tampon is impregnated with the treatment agent (oragents) and that a sufficient amount of the treatment agent (or agents)is present in the tampon.

Typically, the intrauterine device is for placing in the uterus overextended periods of time, such as between one and five years. Typically,the intrauterine device comprises a plastic frame, often in the shape ofa “T” and contains sufficient of the treatment agent(s) to be releasedover the period of use. The agent is generally present within orencompassed by a slow-release polymer which forms part of the device,such as in the form of a “sausage” of agent which wraps around the longarm of the “T” which is typically covered with a controlled-releasemembrane. Intrauterine devices are known in the art.

A second aspect of the invention provides use of at least one agent thatis an antagonist of the IP receptor and/or a PGIS inhibitor, in themanufacture of a medicament for combating a pathological condition ofthe uterus in a female individual.

In an embodiment of this aspect of the invention, the female individualis administered at least one agent that is an antagonist of the IPreceptor and/or a PGIS inhibitor. Typically she is administered the atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor at the same time as the medicament. Alternatively, the femalemay have been (or will be) administered the at least one agent that isan antagonist of the IP receptor and/or a PGIS inhibitor before (orafter) receiving the medicament.

It is appreciated that in this and all subsequent aspects of theinvention, preferences for an agent that is an antagonist of the IPreceptor, and preferences for a PGIS inhibitor, are as describedpreviously with respect to the first aspect of the invention.

It is also appreciated that in this and all subsequent aspects of theinvention, preferences for the pathological condition of the uterus tobe combated are as described previously with respect to the first aspectof the invention.

In an embodiment, the female individual is administered any one or twoor more of an inhibitor of PGES and/or an antagonist of EP2 or EP4, anagent that is an antagonist of the FP receptor, and a COX-2 inhibitor.Typically the female is administered the any one or two or more of theseadditional agents at the same time as the medicament. Alternatively, thefemale may have been (or will be) administered the any one or two ormore of these additional agents before (or after) receiving themedicament containing the at least one agent that is an antagonist ofthe IP receptor and/or a PGIS inhibitor.

Further alternatively, if any two or more of an inhibitor of PGES and/oran antagonist of EP2 or EP4, an agent that is an antagonist of the FPreceptor, and a

COX-2 inhibitor are administered to the female, they may be administeredseparately. Thus, for example, a COX-2 inhibitor may be administeredbefore receiving the medicament, and an inhibitor of PGES and/or anantagonist of EP2 or EP4 may be administered together with themedicament.

It is appreciated that in this and all subsequent aspects of theinvention, preferences for an inhibitor of PGES and/or an antagonist ofEP2 or EP4 are as described previously with respect to the first aspectof the invention.

It is also appreciated that in this and all subsequent aspects of theinvention, preferences for an agent that is an antagonist of the FPreceptor are as described previously with respect to the first aspect ofthe invention.

It is further appreciated that in this and all subsequent aspects of theinvention, preferences for a COX-2 inhibitor are as described previouslywith respect to the first aspect of the invention.

A third aspect of the invention provides use of a combination of atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and an inhibitor of PGES and/or an antagonist of EP2 or EP4,in the manufacture of a medicament for treating or preventing apathological condition of the uterus in a female individual.

In an embodiment, the female individual is administered one or more ofan agent that is an antagonist of the FP receptor, and a COX-2inhibitor. In this case, typically the female is administered one ormore of these additional agents at the same time as the medicament,although the female may have been (or will be) administered one or moreof these additional agents before (or after) receiving the medicament.

A fourth aspect of the invention provides use of a combination of atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and an agent is an antagonist of the FP receptor, in themanufacture of a medicament for treating or preventing a pathologicalcondition of the uterus in a female individual.

In an embodiment, the female individual is administered one or more ofan inhibitor of PGES and/or an antagonist of EP2 or EP4 and a COX-2inhibitor. In this case, typically the female is administered one ormore of these additional agents at the same time as the medicament,although the female may have been (or will be) administered one or moreof these additional agents before (or after) receiving the medicament.

A fifth aspect of the invention provides use of a combination of atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and a COX-2 inhibitor, in the manufacture of a medicament fortreating or preventing a pathological condition of the uterus in afemale individual.

In an embodiment, the female individual is administered one or more ofan inhibitor of PGES and/or an antagonist of EP2 or EP4, and an agentthat is an antagonist of the FP receptor. In this case, typically thefemale is administered one or more of these additional agents at thesame time as the medicament, although the female may have been (or willbe) administered one or more of these additional agents before (orafter) receiving the medicament.

A sixth aspect of the invention provides use of a combination of atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and an inhibitor of PGES and/or an antagonist of EP2 or EP4,and an agent that is an antagonist of the FP receptor, in themanufacture of a medicament for treating or preventing a pathologicalcondition of the uterus in a female individual.

In an embodiment, the female individual is administered a COX-2inhibitor. In this case, typically the female is administered the COX-2inhibitor at the same time as the medicament, although the female mayhave been (or will be) administered the COX-2 inhibitor before (orafter) receiving the medicament.

A seventh aspect of the invention provides use of at least one agentthat is an antagonist of the IP receptor and/or a PGIS inhibitor, and aninhibitor of PGES and/or an antagonist of EP2 or EP4, and a COX-2inhibitor, in the manufacture of a medicament for treating or preventinga pathological condition of the uterus in a female individual.

In an embodiment, the female individual is administered an agent that isan antagonist of the FP receptor. In this case, typically the female isadministered the agent that is an antagonist of the FP receptor at thesame time as the medicament, although the female may have been (or willbe) administered the agent that is an antagonist of the FP receptorbefore (or after) receiving the medicament.

An eighth aspect of the invention provides use of at least one agentthat is an antagonist of the IP receptor and/or a PGIS inhibitor, and anagent that is an antagonist of the FP receptor, and a COX-2 inhibitor,in the manufacture of a medicament for treating or preventing apathological condition of the uterus in a female individual.

In an embodiment, the female individual is administered an inhibitor ofPGES and/or an antagonist of EP2 or EP4. In this case, typically thefemale is administered the inhibitor of PGES and/or an antagonist of EP2or EP4 at the same time as the medicament, although the female may havebeen (or will be) administered the inhibitor of PGES and/or anantagonist of EP2 or EP4 before (or after) receiving the medicament.

A ninth aspect of the invention provides use of a combination of atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, an inhibitor of PGES and/or an antagonist of EP2 or EP4, anagent that is an antagonist of the FP receptor, and a COX-2 inhibitor,in the manufacture of a medicament for treating or preventing apathological condition of the uterus in a female individual.

A tenth aspect of the invention provides use of any one or two or moreof an inhibitor of PGES and/or an antagonist of EP2 or EP4, an agentthat is an antagonist of the FP receptor, and a COX-2 inhibitor, in themanufacture of a medicament for treating or preventing a pathologicalcondition of the uterus in a female individual, wherein the individualis administered at least one agent that is an antagonist of the IPreceptor and/or a PGIS inhibitor. In this case, typically the female isadministered the at least one agent that is an antagonist of the IPreceptor and/or the PGIS inhibitor at the same time as the medicament,although the female may have been (or will be) administered the at leastone agent that is an antagonist of the IP receptor and/or the PGISinhibitor before (or after) receiving the medicament.

In a preferred embodiment of any of the second to tenth aspects of theinvention, the medicament is formulated to be administered via a vaginalring or a tampon or an intrauterine device.

An eleventh aspect of the invention provides a composition comprising atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and any one or two or more of an inhibitor of PGES and/or anantagonist of EP2 or EP4, an agent that is an antagonist of the FPreceptor, and a COX-2 inhibitor.

A twelfth aspect of the invention provides a composition comprising atleast one agent that is an antagonist of the IP receptor and/or a PGISinhibitor, and any one or two or more of an inhibitor of PGES and/or anantagonist of EP2 or EP4, an agent that is an antagonist of the FPreceptor, and a COX-2 inhibitor, for use in medicine.

A thirteenth aspect of the invention provides a pharmaceuticalcomposition comprising at least one agent that is an antagonist of theIP receptor and/or a PGIS inhibitor, and any one or two or more of aninhibitor of PGES and/or an antagonist of EP2 or EP4, an agent that isan antagonist of the FP receptor, and a COX-2 inhibitor, and apharmaceutically acceptable carrier.

A fourteenth aspect of the invention provides a vaginal ring or a tamponor an intrauterine device comprising at least one agent that is anantagonist of the IP receptor and/or a PGIS inhibitor, and any one ortwo or more of an inhibitor of PGES and/or an antagonist of EP2 or EP4,an agent that is an antagonist of the PP receptor, and a COX-2inhibitor.

The invention will now be described in more detail by reference to thefollowing Figures and Examples.

FIG. 1 a: Variation in PGI synthase mRNA expression in endometrialbiopsies from across the menstrual cycle. The relative expression wassignificantly lower in the late proliferative phase of the cycle(1.74±0.51; n=7; p<0.05) compared with the early proliferative phase(10.36±4.58; n=6). Expression was lower in the early secretory(3.56±1.89; n=17; p<0.06) and late secretory (1.42±0.29; n=4; p=0.068)phases.

FIG. 1 b: Temporal variation in IP receptor mRNA expression inendometrial biopsies from across the menstrual cycle. The relativeexpression was significantly lower in the late proliferative phase(2.09±0.57; n=7; p<0.01), early secretory phase (3.16±0.56; n=17;p<0.005) and late secretory phase (2.08±0.55; n=5; p<0.01) compared withthe early proliferative phase (23.28±12.8; n=6).

FIG. 2: Immunohistochemical localisation of PGI synthase infull-thickness endometrial tissue collected in the early proliferative,late proliferative (b) and early secretory (a) phases of the menstrualcycle. Strong staining was seen in the glandular epithelial cells (G) inbasalis (B) and functionalis (F) layers and in the surface epithelium(SE). Stromal staining was seen in the functionalis layer (c) and thebasalis layer (b). Strong staining was present in smooth muscle cells inthe myometrium (Myo) and endothelial reactivity was present in bloodvessels in all layers (arrowed in inset to a, b and c).

FIG. 3: In situ hybridisation of IP receptor probe to sections of humanendometrial tissue collected in the mid proliferative phase of themenstrual cycle. IP receptor was expressed in the myometrium (a) withreactivity present in smooth muscle cells and blood vessels (arrowed).In the basalis layer (b) and the functionalis layer (c and d) somestaining was seen in epithelial cells (G) and in the stroma but stainingwas weaker than that present in the myometrium (M). IP receptorexpression was also present in stromal, cells in the basalis andfunctionalis layers and in endothelial cells in blood vessels (BV)throughout the endometrium (arrowed).

FIG. 4: Cyclic AMP generation in endometrial biopsy tissue in responseto 100 nM iloprost. Incubation with iloprost for 10 min in samplesobtained from the proliferative phase of the menstrual cycle caused a4.83±0.74 fold increase in cAMP generation compared with a 2.07±0.38fold increase from samples obtained from the secretory phase (n=4 foreach group; p<0.05).

FIG. 5: Immunohistochemical localisation of IP receptor in humanendometrial tissue (functional layer and basal-myometrial junction)collected in proliferative phase of the menstrual cycle. Glandularepithelial staining (G) was present in both basal (B) and functional (F)layers and reactivity was detected in smooth muscle cells in themyometrium (M) (FIGS. 5 a-c). Stromal cell staining was presentthroughout the endometrium, but was stronger in the functional layer(FIG. 5 c) compared with the basal layer (FIG. 5 b). Endothelial cellsthroughout the endometrium and at basal-myometrial junction alsoexhibited positive reactivity for IP receptor (indicated by arrows inFIGS. 4 b and c). NEG=Negative control; Scale bars=50 μm.

FIG. 6: Expression and signalling of the IP receptor in Ishikawa and EScells.

FIG. 6 a PCR amplification of a 364 bp fragment of human IP receptor inendometrial tissue (Lane A), ES cells (Lane B) and Ishikawa cells (LaneC). Lane D is a water blank and M is DNA marker.

FIG. 6 b Cyclic AMP generation in Ishikawa and ES cells in response to100 nM iloprost (n=3). Results are expressed as the mean±SEM ofpercentage increase in cAMP induction.

FIG. 7: Proliferation in Ishikawa (n=5) and ES cells (n=6) in responseto treatment with 100 nM iloprost for 24 hours. Results are expressed asthe mean ±SEM percentage increase in proliferation.

FIG. 8: Immunohistochemistry of IP receptor expression in humanendometrium collected from women with excessive (A) or normal (B) bloodloss. Section C is a negative control which is tissue subjected tostaining with DAB. The arrow indicates a blood vessel.

EXAMPLE 1 Temporal Expression, Localisation and Signalling ofProstacyclin (IP) Receptor in the Human Endometrium Across the MenstrualCycle

Abstract

We have studied the site of expression of prostaglandin I synthase(PGIS) and the prostacyclin receptor (IP) in the non-pregnant humanuterus across the menstrual cycle. Using quantitative RT-PCR wedemonstrated significantly increased expression of PGIS (P<0.05) in theearly proliferative compared with the late proliferative phase. Inaddition, IP expression was significantly higher in the earlyproliferative phase of the menstrual cycle compared with the lateproliferative and early and late secretory phases (p<0.01 for allphases). Furthermore, in full thickness human uterine biopsies, PGIS waslocalised by immunohistochemistry to glandular epithelium in basalis andfunctionalis layers, together with stromal cells in both layers.Staining was also seen in endothelial cells, smooth muscle cells andvascular smooth muscle in the myometrium. IP receptor mRNA was detectedby in situ hybridisation, predominantly in proliferative uterus, inmyometrium, blood vessels and endometrial epithelial and stromal cells.The prostacyclin analogue iloprost caused cAMP generation in endometrialtissue, which was significantly increased in samples taken from theproliferative phase of the menstrual cycle compared with the secretoryphase (p<0.05). We have demonstrated up-regulation of PGIS and IP in theearly proliferative phase of the menstrual cycle, with increasedsignalling of the receptor via the cAMP pathway during this phase

Introduction

Prostacyclin (PGI₂) has been characterised as a vasodilator and a potentinhibitor of platelet aggregation (Smyth & Fitzgerald, 2002) and as suchplays an essential role in the maintenance of vascular haemostasis. Itis the main prostanoid synthesised by vascular endothelium and acts as asmooth muscle relaxant. Like other members of the eicosanoid family oflipid mediators cyclcooxygenase (COX) is the key enzyme in the synthesisof PGI₂ from arachidonic acid via PGH₂ the common intermediate inprostaglandin synthesis (Kniss, 1999). There are two major isoforms ofCOX enzymes, COX-1, which is constitutively expressed in many cell typesand COX-2, which is induced by many factors including cytokines andtumour promoters. The specific prostaglandin synthase for PGI₂, PGIsynthase (PGIS), generates PGI₂ from PGH₂. PGI₂ elicits its effects ontarget cells by interaction with its G protein-coupled receptor (IP),which has a typical seven-transmembrane structure (Narumiya et al,1999). The IP receptor can stimulate both Gs and Gq species of Gproteins causing an increase in cAMP generation and inphosphatidylinositol response. PGI₂ has been implicated in the aetiologyof menorrhagia, which is a major problem in women's reproductive health,accounting for 11% of all gynaecological referrals and incurring drugcosts amounting to £7,000,000 in 1995 (Cooper et al, 2001). There isevidence for increased synthesis of PGI₂ (Smith et al, 1981) or anincrease in PGI₂ concentration relative to thromboxane A₂ (Makarainen &Ylikorkala, 1986) in women with menorrhagia compared with controls.However, little information is available on the temporal or spatialexpression of PGIS or IP in the endometrium across the menstrual cycle.

In the present study, we have studied the site of localisation of PGISand IP in non-pregnant endometrium and myometrium by quantitativereverse transcriptase polymerase chain reaction (RT-PCR), in situhybridisation and immunohistochemistry. In addition, we investigated therole of PGI₂ in endometrial cell signalling function by studying theeffect of the prostacyclin analogue iloprost on cAMP generation inendometrial tissue samples.

Materials and Methods

Patients and Tissue Collection

Endometrial biopsies at different stages of the menstrual cycle wereobtained from women with regular menstrual cycles (25-35 days), who hadnot received a hormonal preparation in the three months preceding biopsycollection. Samples were collected either with an endometrial suctioncurette (Pipelle, Laboratoire CCD, France) or as full thicknessendometrial biopsies from women undergoing hysterectomy for benigngynaecological indications. Shortly after collection, tissue was eithersnap frozen in dry ice and stored at −70° C. (for RNA extraction), fixedin neutral buffered formalin (NBF) and wax embedded (forimmunohistochemical analyses), or placed in RPMI 1640 (containing 2 mML-glutamine, 100 U penicillin and 100 μg/ml streptomycin) andtransported to the laboratory for in vitro culture.

Biopsies were dated according to stated last menstrual period (LMP) andconfirmed by histological assessment according to criteria of Noyes andco-workers (Noyes et al, 1975). Furthermore, circulating oestradiol andprogesterone concentrations at the time of biopsy were consistent forboth stated LMP and histological assignment of menstrual cycle stage.Ethical approval was obtained from Lothian Research Ethics Committee andwritten informed consent was obtained from all subjects before tissuecollection.

Taqman Quantitative RT-PCR

RNA was extracted from endometrial biopsies obtained from across themenstrual cycle (n=35) using Tri-Reagent (Sigma, Poole, UK) followingthe Manufacturer's instructions. RNA samples were quantified and werereverse transcribed using 5.5 mM MgCl₂, 0.5 mM each deoxy (d)-NTPs, 2.5μM random hexamers, ribonuclease inhibitor (0.4 U/μl) and 1.25 U/μlMultiscribe reverse transcriptase (all from Applied Biosystems,Warrington, UK). RNA (400 ng) was added to each reverse transcriptionreaction and samples were incubated for 90 min. at 25° C., 45 min. at48° C. and 5 min. at 95° C. The reaction mix for the polymerase chainreaction (PCR) consisted of 1× mastermix, ribosomal 18S forward andreverse primers, ribosomal 18S probe (50 nM; all from ABI), forward andreverse primers for PGIS or IP (300 nM) and PGIS or IP probe (200 nM)(all from Biosource UK Ltd). The reaction mix (48 μl) was aliquoted intotubes and 2 μl cDNA was added. Duplicate 24 μl samples plus positive andnegative controls were placed in a PCR plate and wells were sealed withoptical caps. The PCR reactions were carried out using an ABI Prism 7700(Applied Biosystems). All primers and probe were designed using thePRIMER express program (ABI).

The sequences of PGIS primers and probe were: forward,5′-ACGCAGATGTGGAGATCCCT-3′ (SEQ ID No 27); reverse,5′-GTCGTGTTCCGGCTGCA-3′ (SEQ ED No 28); and probe (6-carboxy fluorosceinlabelled) 5′-CCTCAGCAGGTACGGCTTCGGTCTG-3′ (SEQ ID No 29).

The sequences of the IP primers and probe were: forward,5′-GCCCTCCCCCTCTACCAA-3′ (SEQ ID No 30); reverse,5′-TTTTCCAATAACTGTGGTTTTTGTG-3′ (SEQ ID No 31); and probe (6-carboxyfluoroscein labelled) 5′-CCAAGAGCCAGCCCCCTTTCTGC-3′ (SEQ ID No 32).

The sequences of 18S primers and probe have been described previously(Milne et al, 2001).

Data were analysed and processed using Sequence Detector version 1.6.3(ABI) according to manufacturer's instructions. Results were expressedrelative to an internal positive standard included in all reactions.

Non-Quantitative RT-PCR

RNA (1-2 μg per sample) was reverse transcribed using 4 U per reactionof Omniscript reverse transcriptase (Qiagen, Crawley, UK) in 1× reactionbuffer containing 0.5 mM of each DNTP, 50 ng/μl Oligo-dT primer and 10ng/μl random hexamers in a volume of 20 μl. Reverse transcription wasfor 1 hour at 37° C. followed by 2 minutes at 93° C. A 364 bp fragmentof human IP receptor mRNA from base 672 to 1035 was amplified usingprimers forward, 5′-CAACGGCTCGGTCACCCTCAGC-3′ (SEQ ID No 33) andreverse, 5′-AAGGGGTGTCTGCGAGTCTCCG-3′ (SEQ ID No 34) and HotStarTaq DNApolymerase (Qiagen, Crawley, UK) according to manufacturer'sinstructions. Five microlitres of each cDNA was added to 2.5 U enzyme,200 μM of each dNTP and 100 ng of each primer in 50 ml of 1× reactionbuffer. Amplification consisted of 1 cycle of 95° C. for 15 minutes, 40cycles of denaturation at 95° C. for 1 minute, annealing at 66° C. for30 seconds and extension at 72° C. for 45 seconds. This was followed bya final extension at 72° C. for 10 minutes. Reactions were resolved on a1% agarose gel and visualised by ethidium bromide transillumination.Sequences of representative pcr products were confirmed by customsequencing (MWG-Biotech AG, Ebersberg, Germany).

In Situ Hybridisation

A custom synthesised oligonucleotide double fluoroscein isothiocyanate(FITC)-labelled cDNA probe for IP was obtained from Biognostik(Gottingen, Germany). Sections (5 μM) from full thickness human uterinebiopsies collected across the menstrual cycle (n=12) were cut ontogelatin-coated slides. Sections were dewaxed and rehydrated and thentreated with proteinase K (50 μg/ml in 100 mM Tris-HCl pH 7.6,containing 50 mM EDTA) for 15 mins at 37° C. to enhance cDNA probeaccess. Sections were washed in diethylpyrocarbonate treated water andpre-hybridised for 4 hours at 30° C. with 25 μl of the hybridisationbuffer supplied with the probe, which had been previously heated to 95°C. The sections were then hybridised overnight at 30° C. with the cDNAprobe at 6 U/ml in hybridisation buffer. Following hybridisation,sections were washed for 2×5 min. in 1× standard saline citrate (SSC)and 2×15 min. in 0.1×SSC at 39° C. The FITC-labelled probe was detectedusing standard immununohistochemical reagents with an additionalamplification step (TSA Biotin System, NEN Life Sciences, UK). Sectionswere incubated with blocking buffer for 30 min. Conjugatedanti-PITC-horseradish peroxidase (Roche, Diagnostics Ltd., Lewes, UK)was added at a dilution of 1 in 200 in blocking buffer and the sectionsincubated for 60 min. After washing, biotinyl tyramide amplificationreagent (1 in 50) was applied to each slide and incubated for 15 min.Streptavidin-horseradish peroxidase (1 in 100) was applied after washingand incubated for 30 min. and probe localisation visualised with3,3′-diaminobenzidine (DAB) substrate. Control sections were treatedwith a double FITC-labelled oligonucleotide probe containing the sameproportion of cysteine (C) and guanine (G) bases as the IP probe toassess background hybridization. All treatments were carried out at roomtemperature unless otherwise specified.

Immunohistochemistry

Endometrial sections (5 μm) from across the menstrual cycle (n=12) weredewaxed in xylene and rehydrated using decreasing grades of ethanol.Antigen retrieval was performed by treating sections for 5 min. in apressure cooker in boiling 0.1% citrate buffer, pH 3.0. After rinsing inPBS, endogenous peroxidase activity was quenched with 10% H₂O₂ inmethanol at room temperature. Non-immune swine serum (20% serum in PBS)was applied for 1 hour before overnight incubation at 4° C. with arabbit anti-bovine PGIS. An avidin-biotin peroxidase detection systemwas then applied (DAKO Ltd., Cambridge, UK) with DAB as the chromagen.The anti-PGIS antibody was obtained from Alexis Corporation (Nottingham,UK).

An avidin-biotin peroxidase detection system was then applied (DAKOLtd., Cambridge, UTK) with DAB as the chromagen. The antibody to IPreceptor has been described previously (Fortier et al (2001)Prostaglandins Leukot Essent Fatty Acids 65, 79-83). Non-immune rabbitserum and antibody pre-absorbed with IP receptor peptide were used ascontrols for IP receptor and non-immune rabbit serum was used as controlfor PGIS. Immunoreactivity was negligible with pre-absorbed antibody andwith non-immune rabbit serum there was occasional generalised pale browncross reactivity over epithelial glands and endothelium.

Whole Tissue Cyclic AMP Assay

Endometrial biopsies from across the menstrual cycle (n=8) were mincedfinely with scissors and divided into three portions. The tissue wasincubated is overnight at 37° C. in a humidified 5% CO₂ incubator in 2ml RPMI (Sigma, UK) medium containing, 2 mmol” L-glutamine, 100 IUpenicillin and 100 μg streptomycin and 3 μg/ml indomethacin (Sigma,Poole, UK). Following overnight incubation, the tissue was incubated inthe same medium containing 1-methyl-3-isobutylxanthine (Sigma) at 37° C.for 30 min. It was then treated with control medium or 100 nM iloprost(a gift from Schering Health Care, Burgess Hill, UK) for 10 min. at 37°C. and lysed in 0.1M HCl and frozen until assayed. cAMP concentrationwas measured by ELISA (Biomol, Affiniti, Exeter, UK) according to themanufacturer's instructions and normalised to protein concentrationdetermined by a modification of the method of Lowry (Bio-Rad, HemelHempstead, UK).

Cyclic AMP and Proliferation Assays in Cell Lines

The Ishikawa endometrial epithelial cell line (Ishikawa cells) andendometrial stromal (ES) cells were propagated in DMEM F12 culturemedium with glutamax (Invitrogen Ltd., Paisley, UK) supplemented with10% foetal bovine serum (PAA Laboratories Ltd., Yeovil, UK). Endometrialstromal cells were a generous gift from Professor R W Kelly and theywere derived from human endometrial biopsy tissue as describedpreviously (Dunn et al (2002) J Clin Endocrinol Metab 87, 1898-1901).For cyclic AMP assay, cells were grown in 6 well plates to 70%confluence. They were starved overnight in serum free medium in thepresence of indomethacin (3 μg/ml). Cells were then incubated in thesame medium containing 1-methyl-3-isobutylxanthine at 37° C. for 30minutes and treated with control medium or 100 nM iloprost for 10minutes at 37° C. Following treatment, they were lysed in 0.1M HCl andfrozen until assayed. The cyclic AMP assay was performed as for tissuesamples. Data are presented as mean±SEM percentage increase in cAMPafter treatment with iloprost, where the increase was calculatedrelative to the control samples. To assess the effect of iloprost onproliferation, cells were seeded in 96 well plates and grown to 70%confluence. They were starved overnight in serum free medium in thepresence of indomethacin (3 μg/ml). Cells were treated with controlmedium or iloprost at a concentration of 100 nM (6 wells for eachtreatment; data presented as mean of 5 or 6 experiments) for 24 hours at37° C. Proliferation was assessed using a colorimetric method, theCellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega,Southampton, UK) according to manufacturer's instructions. Twentymicrolitres of the assay reagent (containing a tetrazolium compound,inner salt and an electron coupling reagent) were added to each well andsamples were incubated at 37° C. for 2 hours and plates were read at 492nm. Proliferation is presented as percentage increase relative tountreated cells and plotted as the mean±SEM.

Statistics

Where appropriate, data were subjected to statistical analysis withANOVA and Fishers PLSD tests (Statview 4.0; Abacus Concepts Inc., USA)and statistical significance accepted when p<0.05.

Results

The pattern of PGIS mRNA expression in the human endometrium across themenstrual cycle was studied by quantitative RT-PCR. PGIS mRNA wasdetected in all samples of human endometrium examined (FIG. 1 a).Relative expression was higher (p<0.05) in the early proliferative phaseof the menstrual cycle (10.36±4.58; n=6) than in the late proliferativephase (1.74±0.51; n=7;). PGIS expression was low in the early secretory(3.56+1.89; n=17) or late lo secretory (1.42+0.29; n=4) phases of themenstrual cycle.

The temporal pattern of IP receptor mRNA expression across the menstrualcycle was also assessed by quantitative RT-PCR. IP mRNA was detected inall samples of human endometrium examined by RT-PCR (FIG. 1 b). However,the relative expression was significantly higher in the earlyproliferative phase of is the menstrual cycle (23.28±12.8; n=6) comparedwith the late proliferative (2.09±0.57; n=7; p<0.01), early secretory(3.16±0.56;n=17; p<0.01) or late secretory (2.08±0.55; n−5; p<0.01)phases.

Immunohistochemical staining for PGIS was performed in full thicknesshuman uterine biopsies obtained from across the menstrual cycle.Cytoplasmic and nuclear staining were present in glandular epithelialcells in the basalis (FIG. 2 b) and functionalis FIG. 2 c) layers.Stromal cell reactivity was also present in both layers, together withendothelial cell staining in the microvasculature (arrowed in FIG. 2).Myometrial smooth muscle cells showed both cytoplasmic and nuclearreactivity (Inset to FIG. 2 a).

The site of expression of IP mRNA was studied by in situ hybridisation.IP reactivity was detected in uterine samples collected during theproliferative phase of the menstrual cycle and was localised inmyometrial smooth muscle cells (FIGS. 3 a and b) and in endothelialcells lining vessels in all uterine layers (FIGS. 3 a and c). Expressionwas also present in glandular epithelial cells of the basalis andfunctionalis layers (FIGS. 3 b-d), mainly in the cytoplasm, but alsooverlying nuclei. Stromal cell expression was present in the basalis andfunctionalis regions (FIGS. 3 b-d).

Immunohistochemical staining for IP receptor protein was detectedthroughout the menstrual cycle in the cytoplasm and nuclei of glandularepithelial cells in both basal and functional layers (FIGS. 5 a-c).Similar to PGIS immunoreactivity, IP receptor stromal cell staining wasstronger and more widespread in the functional layer (FIG. 5 c) comparedwith the basal layer (FIG. 5 b). IP receptor protein expression was alsolocalised to endothelial cells throughout the microvasculature and waspresent in myometrial smooth muscle cells.

To investigate signalling via the IP receptor, cAMP generation inresponse to iloprost treatment was assessed in endometrial biopsy tissue(FIG. 4). In biopsy samples, cAMP generation in response to iloprost wassignificantly higher in endometrial samples collected during theproliferative phase compared with the secretory phase (4.83±0.74 vs2.07±0.39; n=4 for each group; p<0.05).

To investigate the potential role of the IP receptor in proliferation ofepithelial and stromal cells of the endometrium, Ishikawa and ES cellswere assessed for expression and signalling of the IP receptor and thenutilised for further functional studies. Amplification and sequencing ofa 364 bp fragment of IP receptor by conventional techniques confirmedexpression of IP receptor in both cell types (FIG. 6 a). Moreover, cAMPgeneration in Ishikawa and ES cells was significantly elevated followingtreatment with 100 nM iloprost (166%±27.6% and 37,936%±18,464% ofcontrol values respectively; n=3; p<0.05) (FIG. 6 b).

Finally, the effect of iloprost on proliferation was assessed in theIshikawa and ES cells (FIG. 7). Treatment with iloprost resulted in asignificant increase in proliferation in both Ishikawa and ES cells(109.4%±2.4% and 112%±3.8% of control values respectively; p<0.05).

Discussion

This study demonstrates the expression of PGIS and IP receptor genes inthe human endometrium and shows significant upregulation of both duringmenstruation. PGIS is the terminal enzyme that leads to synthesis ofPGI₂ in target tissue (Kniss, D. A. (1999) J Soc Gynecol Investig 6,285-292). The higher expression level of PGIS during menstruationsupports previous observations reporting temporal pattern of PGI₂secretion by the human endometrium across the menstrual cycle; PGI₂concentrations are maximal in uterine venous blood during menstruation(Goodfellow et al (1982) Thromb Haemost 48, 9-12). The elevatedexpression of IP receptor during menstruation suggests that PGI2synthesis and IP receptor are temporally regulated to induce theireffects on target cells. This is supported by the maximal Iloprostinduced cAMP response observed in endometrium collected during themenstrual/proliferative phases. The IP receptor has been shown to coupleto G_(s) proteins which are linked to increase in cAMP generation(Narumiya et at (1999) Physiol Rev 79, 1193-1226). Although only theprotein kinase A pathway was investigated in this study, it is predictedthat the IP receptor may activate other signalling pathways in the humanendometrium as has been shown recently for other prostaglandin receptors(Milne et al (2003) J Clin Endocrinol Metab 88, 1825-1832; Jabbour et al(2003) J Soc Gynaecol Invest (Supplement) 10, 75). Such diversesignalling pathways may lead to differential activation of target genesthat can promote various phenotypic changes on target cells.

The factors that regulate the expression of the PGIS and IP receptorduring menstruation in the human endometrium are not clear. It is likelythat this temporal expression is regulated by steroid hormones as hasbeen postulated for other prostanoids and their receptors (Milne et al(2001) J Clin Endocriizol Metab 86, 4453-4459; Milne et al (2003) J ClinEndocrinol Metab 88, 1825-1832). Oestradiol-17β has been shown tostimulate the secretion of PGI₂ in endometrial stromal cells (Levin etal (1992) Fertil Steril 58, 530-536). Whether this is associated withup-regulation in expression of IP receptor is unclear. Expression of theIP receptor may be regulated also by PGI₂ or other prostaglandins.Expression of PGIS and synthesis of PGI₂ is induced by COX-2 (Caughey etal (2001) J Immunol 167, 2831-2838), which is up-regulated during thetime of menstruation (Jones et al (1997) Hum Reprod 12, 1300-1306). Itis also plausible that local mediators within the endometrium may play arole in regulation of PGI₂ synthesis and/or expression of its receptor.For instance, prostacyclin is a mediator of the protective effects ofVEGF on the vasculature (Zachary, I. (2001) Am J Physiol Cell Physiol280, C1375-1386) and PGI₂ biosynthesis is upregulated by VEGF via ERKmediated cPLA₂ activation and arachidonic acid mobilisation (Zachary &Gliki (2001) Cardiovasc Res 49, 568-581).

PGIS and IP receptor expression have been co-localised to multi-cellularcompartments of the human endometrium. These include stromal, glandularepithelial, endothelial and smooth muscle cells. This suggests thatprostacyclin acts in an autocrine/paracrine manner within the humanendometrium to induce its cellular effects. PGIS and IP receptorimmunoreactivities have been demonstrated previously in myocytes,vascular smooth muscle cells and endothelial cells in pregnant andnon-pregnant human myometrium (Moonen et al (1986) Br J Obstet Gynaecol93, 255-259; Chegini & Rao (1988) J Clin Endocrinol Metab 66, 76-87;Giannoulias et al (2002) J Clin Endocrinol Metab 87, 5274-5282). To ourknowledge, however this the first report of localisation of PGIS and IPreceptor in glandular epithelial and stromal cells within the humanendometrium. Previous studies using autoradiography with ³[H] PGI₂ onhuman uterine tissue failed to demonstrate PGI₂ binding sites inepithelial cells (Chegini & Rao (1988) J Clin Endocrinol Metab 66,76-87), although in that study binding sites were demonstrated inmyometrial smooth muscle. This inconsistency with findings presentedherein may reflect differences in the sensitivity of the methods used.Interestingly, stromal expression of PGIS and IP receptor was highest inthe functional layer of the endometrium. In pre-menopausal women, thehuman endometrium undergoes phases of proliferation and apoptosis duringsuccessive menstrual cycles. These phases are observed predominantly inthe functional layer of the endometrium, which is shed at menstruationbefore regenerating during the proliferative phase of the subsequentmenstrual cycle. Hence this spatio-temporal expression of PGIS and IPreceptor may be crucial for, and in keeping with, its predicted role inmenstruation.

Baird et al ((1996) Eur J Obstet Gynecol Reprod Biol 70, 15-17), havepostulated a role for PGI₂ in menstruation based on its myometrialsmooth muscle and vascular relaxation effects and inhibition of plateletaggregation. This would counteract the effects of other prostaglandinssuch as PGF_(2α), which causes vasoconstriction and myometrial smoothmuscle contraction (Crankshaw & Dyal (1994) Can J Physiol Pharmacol 72,870-874). It is also likely that PGI₂ is involved in the repair of thevascular bed, since it has protective effects on the endothelium byinhibiting vascular smooth muscle proliferation and enhancingendothelial cell survival (Zachary, I. (2001) Am J Physiol Cell Physiol280, C1375-1386). The increased expression of PGIS and IP receptorduring menstruation is also consistent with a role for PGI₂ in theaetiology of menorrhagia. Evidence for this has been provided previouslyby studies of dysfunctional menstrual bleeding, which have demonstratedincreased synthesis of PGI₂ (Smith et al (1981) Lancet 1, 522-524) orincreased synthesis of PGI₂ relative to thromboxane A₂ (Makarainen &Ylikorkala (1986) Br J Obstet Gynaecol 93, 974-978) in uterine tissuefrom women with excessive blood loss relative to controls. Whether IPreceptor expression and signalling are also elevated in endometrium ofwomen with dysfunctional menstruation remains to be established.

The role of prostacyclin in endometrial stromal and epithelial cells isunclear. These cells were shown to express functional IP receptors andtreatment of both cell types with Iloprost resulted in modest increasesin cell proliferation. Prostacyclin has been shown to have bothinhibitory and stimulatory effects on proliferation depending on thecell type (Clapp et al (2002) Am J Respir Cell Mol Biol 26, 194-201;Murphy & Fitzgerald (2001) Faseb J 15, 1667-1669). However, the Iloprostinduced proliferation observed in endometrial epithelial and stromalcells are much lower than what is previously reported with otherprostaglandins such as PGE₂ (25% increase) (Jabbour & Boddy (2003) J SocGynaecol Invest (Supplement) 10, 75 and PGF_(2α) (31% increase) (Milne &Jabbour (2003) J Clin Endocrinol Metab 88, 1825-1832). This differenceis reflected in the temporal pattern of expression of the IP receptorcompared with the EP and FP receptors. Expression of IP receptor ishighest during menstruation whereas expression of receptors for PGE₂(namely EP4) and PGF_(2α) (FP) are detected maximally during themid-late proliferative phase (Milne et al (2001) J Clin Endocrinol Metab88, 4453-4459; Milne & Jabbour (2003) J Clin Endocninol Metab 88,1825-1832). It is tempting to speculate that expression of IP receptorsin stromal and epithelial cells may induce genes that are involved invascular function of the endometrium during menstruation. Expression ofa number of these genes such as VEGF, angiopoietins, bFGF or nitricoxide are expressed in the human glandular and/or stromal cells of theendometrium (Gargett & Rogers (2001) Reproduction 121, 181-186; Hewettet al (2002) Am J Pathol 160, 773-780).

In summary, this study has demonstrated temporal expression of PGIS andIP receptor in the non-pregnant human endometrium across the menstrualcycle. Expression of both genes is highest during the menstrual phaseand is localised to multi-cellular compartments within the endometriumand myometrium. The function of prostacyclin in the human endometrium islinked to protein kinase A pathway during menstruation. Moreover,treatment of endometrial stromal and epithelial cells with aprostacyclin analogue induces modest increases in proliferation. Futurestudies will elucidate the role of prostacyclin in menstruation and themechanisms of its signalling in the human endometrium.

EXAMPLE 2 Expression of IP Receptors in Uterine Tissue of Women WithMenorrhagia Compared to Women With No Menorrhagia

Endometrial tissue was collected by biopsy from a woman with a knownindication of menorrhagia and and a woman who have normal uterinefunction during the proliferative phase of the menstrual cycle. Thetissue was assessed by immunohistochemistry using the method describedin Example 1 using the IP receptor antibody (Fortier et al (2001)Prostaglandins Leukol Essent Fatty Acids 65, 79-83) at a dilution of1/500.

As shown in FIG. 8, expression of the IP receptor is elevated inendometrial tissue from a woman with a known history of menorrhagia (A)compared to a woman with normal blood loss (B).

There is both vascular and glandular epithelial cell staining in theendometrium. Staining m blood vessels suggests that prostacyclin has adirect effect on the versel function such as increased permeability ofthe vasculature which may lead to increased blood leakage.

Hence, in women with menorrhagia it should prove beneficial to treatwith IP receptor antagonists, in order to block the signalling pathwayand ultimately transcription of target genes that may mediate vascularfunction/dysfunction and excessive bleeding.

EXAMPLE 3 Treatment of Uterine Cancer With IP Receptor Antagonist

A patient suffering from uterine cancer is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid at a dosing quantity and frequency such that the therapeutic levelof active agent at the site of treatment is maintained at a levelideally EC90 but preferably not less than EC50 throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 4 Treatment of Uterine Cancer With an IP Receptor Antagonist andan EP2 Receptor Antagonist

A patient suffering from uterine cancer is administered4(4-{3-[4(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester or 2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline andAH6809 at a dosing quantity and frequency such that the therapeuticlevel of active agent at the site of treatment is maintained at a levelideally EC90 but preferably not less than EC50 throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 5 Treatment of Uterine Cancer With an IP Receptor Antagonist andan EP4 Receptor Antagonist.

A patient suffering from uterine cancer is administered(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid or 3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinicacid and AH23848B at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 6 Treatment of Uterine Cancer With an IP Receptor Antagonist andan FP Receptor Antagonist

A patient suffering from uterine cancer is administered2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester and AL-3138 or AL-8810 at a dosing quantity andfrequency such that the therapeutic level of each active agent at thesite of treatment is maintained at a level ideally EC90 but preferablynot less than EC50 throughout the treatment period. The treatment isdelivered orally or more locally depending on patient acceptability,avoidance of side effects and systemic bioavailability.

EXAMPLE 7 Treatment of Uterine Cancer With an IP Receptor Antagonist anda COX-2 Inhibitor

A patient suffering from uterine cancer is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or(R)-3-(1H-benzdimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and nimesulide at a dosing quantity and frequency such that thetherapeutic level of active agent at the site of treatment is maintainedat a level ideally EC90 but preferably not less than EC50 throughout thetreatment period. The treatment is delivered orally or more locallydepending on patient acceptability, avoidance of side effects andsystemic bioavailability.

EXAMPLE 8 Treatment of Fibroids With an IP Receptor Antagonist

A patient suffering from fibroids is administered4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid at a dosing quantity and frequency such that the therapeutic levelof active agent at the site of treatment is maintained at a levelideally EC90 but preferably not less than EC50 throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 9 Treatment of Fibroids With an IP Receptor Antagonist and anEP2 Receptor Antagonist

A patient suffering from fibroids is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline and AH6809 at a dosingquantity and frequency such that the therapeutic level of each activeagent at the site of treatment is maintained at a level ideally EC90 butpreferably not less than EC50 throughout the treatment period. Thetreatment is delivered orally or more locally depending on patientacceptability, avoidance of side effects and systemic bioavailability.

EXAMPLE 10 Treatment of Fibroids With an IP Receptor Antagonist and anEP4 Receptor Antagonist

A patient suffering from fibroids is administered(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid or 3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinicacid and AH22921 at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 11 Treatment of Fibroids With an IP Receptor Antagonist and anFP Receptor Antagonist

A patient suffering from fibroids is administered2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and AL-3 138 or AL-8810 at a dosing quantity and frequency suchthat the therapeutic level of each active agent at the site of treatmentis maintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 12 Treatment of Fibroids With an IP Receptor Antagonist and aCOX-2 Inhibitor

A patient suffering from fibroids is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylnethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid and nimesulide at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 13 Treatment of Endometriosis With an IP Receptor Antagonist

A patient suffering from endometriosis is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or4-(4-(3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester at a dosing quantity and frequency such that thetherapeutic level of active agent at the site of treatment is maintainedat a level ideally EC90 but preferably not less than EC50 throughout thetreatment period. The treatment is delivered orally or more locallydepending on patient acceptability, avoidance of side effects andsystemic bioavailability.

EXAMPLE 14 Treatment of Endometriosis With an IP Receptor Antagonist andan EP2 Receptor Antagonist

A patient suffering from endometriosis is administered4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and AH6809 at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 15 Treatment of Endometriosis With an IP Receptor Antagonist andan EP4 Receptor Antagonist

A patient suffering from endometriosis is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid andAH2291 at a dosing quantity and frequency such that the therapeuticlevel of each active agent at the site of treatment is maintained at alevel ideally EC90 but preferably not less than EC50 throughout thetreatment period. The treatment is delivered orally or more locallydepending on patient acceptability, avoidance of side effects andsystemic bioavailability.

EXAMPLE 16 Treatment of Endometriosis With an IP Receptor Antagonist andan FP Receptor Antagonist

A patient suffering from endometriosis is administered2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or 2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline and AL-3138 orAL-8810 at a dosing quantity and frequency such that the therapeuticlevel of each active agent at the site of treatment is maintained at alevel ideally EC90 but preferably not less than EC50 throughout thetreatment period. The treatment is delivered orally or more locallydepending on patient acceptability, avoidance of side effects andsystemic bioavailability.

Example 17 Treatment of Endometriosis With an IP Receptor Antagonist anda COX-2 Inhibitor.

A patient suffering from endometriosis is administered4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and nimesulide at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 18 Treatment of Menorrhagia With an IP Receptor Antagonist

A patient suffering from endometriosis is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid at a dosing quantity and frequency such that the therapeutic levelof active agent at the site of treatment is maintained at a levelideally EC90 but preferably not less than EC50 throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 19 Treatment of Menorrhagia With an IP Receptor Antagonist andan EP2 Receptor Antagonist

A patient suffering from endometriosis is administered4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester or 2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline andAH6809 at a dosing quantity and frequency such that the therapeuticlevel of each active agent at the site of treatment is maintained at alevel ideally EC90 but preferably not less than EC50 throughout thetreatment period. The treatment is delivered orally or more locallydepending on patient acceptability, avoidance of side effects andsystemic bioavailability.

EXAMPLE 20 Treatment of Menorrhagia With an IP Receptor Antagonist andan EP4 Receptor Antagonist

A patient suffering from endometriosis is administered(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid or 3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinicacid and AH2291 at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 21 Treatment of Menorrhagia With an IP Receptor Antagonist andan FP Receptor Antagonist

A patient suffering from endometriosis is administered2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester and AL-3 138 or AL-8810 at a dosing quantity andfrequency such that the therapeutic level of each active agent at thesite of treatment is maintained at a level ideally EC90 but preferablynot less than EC50 throughout the treatment period. The treatment isdelivered orally or more locally depending on patient acceptability,avoidance of side effects and systemic bioavailability.

EXAMPLE 22 Treatment of Menorrhagia With an IP Receptor Antagonist and aCOX-2 Inhibitor

A patient suffering from endometriosis is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and nimesulide at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 23 Treatment of Dysmenorrhoea With an IP Receptor Antagonist

A patient suffering from endometriosis is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid at a dosing quantity and frequency such that the therapeutic levelof active agent at the site of treatment is maintained at a levelideally EC90 but preferably not less than EC50 throughout the treatmentperiod. The treatment is delivered orally or more locally depending onpatient acceptability, avoidance of side effects and systemicbioavailability.

EXAMPLE 24 Treatment of Dysmenorrhoea With an IP Receptor Antagonist andan EP2 Receptor Antagonist

A patient suffering from endometriosis is administered2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or4-(4-{3-[4-(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester and AH6809 at a dosing quantity and frequency such thatthe therapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 25 Treatment of Dysmenorrhoea With an IP Receptor Antagonist andan EP4 Receptor Antagonist

A patient suffering from endometriosis is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and AH2291 at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

EXAMPLE 26 Treatment of Dysmenorrhoea With an IP Receptor Antagonist andan FP Receptor Antagonist

A patient suffering from endometriosis is administered3-(5-phenyl-benzofuran-2-ylmethoxycarbonyl-amino)-isonicotinic acid or2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-5-methanesulfonylamino-benzoicacid or4-(4-{3-[4(4,5-Dihydro-1H-imidazol-2-ylamino)-phenyl]-propionyl}-3-fluoro-phenyl)-piperazine-1-carboxylicacid ethyl ester and AL-3138 or AL-8810 at a dosing quantity andfrequency such that the therapeutic level of each active agent at thesite of treatment is maintained at a level ideally EC90 but preferably20 not less than EC50 throughout the treatment period. The treatment isdelivered orally or more locally depending on patient acceptability,avoidance of side effects and systemic bioavailability.

EXAMPLE 27 Treatment of Dysmenorrhoea With an IP Receptor Antagonist anda COX-2 Inhibitor

A patient suffering from endometriosis is administered2-[4-(4-isoproxybenzyl)phenyl]amino-imidazoline or(R)-3-(1H-benzoimidazol-2-yl)-2-(biphenyl-4-ylmethoxycarbonylamino)-propionicacid and nimesulide at a dosing quantity and frequency such that thetherapeutic level of each active agent at the site of treatment ismaintained at a level ideally EC90 but preferably not less than EC50throughout the treatment period. The treatment is delivered orally ormore locally depending on patient acceptability, avoidance of sideeffects and systemic bioavailability.

REFERENCES

-   Smyth E M, FitzGerald G A (2002) Human prostacyclin receptor. Vitam    Horm 65: 149-165.-   Kniss D A (1999) Cyclooxygenases in reproductive medicine and    biology. J Soc Gynecol Investig 6: 285-292.-   Narwniya S, Sugimoto Y, Ushikubi F (1999) Prostanoid receptors:    structures, properties, and functions. Physiol Rev 79: 1193-1226.-   Cooper K G, Jack S A, Parkin D E, Grant A M (2001) Five-year follow    up of women randomised to medical management or transcervical    resection of the endometrium for heavy menstrual loss: clinical and    quality of life outcomes. Bjog 108: 1222-1228.-   Smith S K, Abel M H, Kelly R W, Baird D T (1981) A role for    prostacyclin (PGi2) in excessive menstrual bleeding. Lancet 1:    522-524.-   Makarainen L, Ylikorkala O 1986 Primary and myoma-associated    menorrhagia: role of prostaglandins and effects of ibuprofen. Br J    Obstet Gynaecol 93: 974-978.-   Noyes R W, Hertig A T, Rock J 1975 Dating the endometrial biopsy. Am    J Obstet Gynecol 122: 262-263.-   Milne S A, Perchick G B, Boddy S C, Jabbour H N (2001) Expression,    localization, and signaling of PGE(2) and EP2/EP4 receptors in human    nonpregnant endometrium across the menstrual cycle. J Clin    Endocrinol Metab 86: 4453-4459.-   Ashby, B. (1998) Co-expression of prostaglandin receptors with    opposite effects: a model for homeostatic control of autocrine and    paracrine signalling. Biochem Pharmacol 55: 239-246.-   Coleman, R A, Smiith, W L, Narumiya, S. (1994) International Union    of Phannacology classification of prostanoid receptors: properties,    distribution, and structure of the receptors and their subtypes.    Pharmacol Rev 46: 205-229.-   DeWitt, D L. (1991) Prostaglandin endoperoxide synthase: regulation    of enzyme expression. Biochim Biophys Acta 1083: 121-134.-   Gordon, M D, Ireland, K. (1994) Pathology of hyperplasia and    carcinoma of the endometrium. Semin Oncol 21: 64-70.-   Herschman, H R. (1996) Prostaglandin synthase 2. Biochlim Biophys    Acta 1299: 125-140.-   Mant, J W, Vessey, M P. (1994) Ovarian and endometrial cancers.    Cancer Surv 20: 287-307.-   Subbaramaiah, K, Telang, N, Ramonetti, J T, Araki, R, DeVito, B,    Weksler, B B, Dannenberg, A J. (1996) Transcription of    cyclooxygenase-2 is enhanced in transformed mammary epithelial    cells. Cancer Res 56: 4424-4429.

1. A method of preventing or treating a pathological condition of theuterus in a female individual, the method comprising administering tothe individual at least one agent that is an antagonist of the IPreceptor and/or an inhibitor of prostaglandin I synthase (PGIS).
 2. Themethod according to claim 1 wherein the pathological condition of theuterus is associated with abnormal growth of cells of the myometrium orendometrium.
 3. The method according to claim 1 wherein the pathologicalcondition of the uterus is selected from uterine carcinoma, menorrhagia,dysmenorrhoea and an endometrial or myometrial pathological condition.4. The method according to claim 3 wherein the endometrial pathologicalcondition is endometriosis.
 5. The method according to claim 3 whereinthe myometrial pathological condition is fibroids.
 6. The methodaccording to any of claim 1 wherein the antagonist of the IP receptorprevents or reduces the binding of PGI2 to the IP receptor.
 7. Themethod according to any of claim 1 wherein the antagonist of the IPreceptor affects the interaction between PGI2 and the IP receptor, orthe interaction between the IP receptor and the associated G protein,thus inhibiting or disrupting a PGI2-IP mediated signal transductionpathway.
 8. The method according to claim 1 wherein the IP receptorantagonist is any one or more of a 2-(arylphenyl)amino-imidazolinederivative a 2-(substituted-phenyl)amino-imidazoline derivative analkoxycarbonylamino heteroaryl carboxylic acid derivative analkoxycarbonylamino benzoic acid or alkoxycarbonylamino tetrazolylphenyl derivative a 2-phenylaminoimidazoline phenyl ketone derivative acarboxylic acid derivative an amino- or amido-prostacyclin derivativecompound a 15(R)-isocarbacyclin or 15-deoxyisocarbacyclin derivative a6,9-thiaprostacyclin analogue or derivative (5Z)-carbacyclin; FCE 22176((5Z)-13,14-didehydro-20-methyl-carboprostacyclin); and an anti-IPreceptor antibody.
 9. The method according to claim 1 wherein the agentis an antagonist of PGI2.
 10. The method according to claim 1 whereinthe agent is an inhibitor of PGIS.
 11. The method according to claim 10wherein the PGIS inhibitor is an anti-PGIS antibody, U-51605,peryoxynitrite, 3-morpholinosydnonimine N-ethylcarbamide ortrans-2-phenylcyclopropylamine HCl.
 12. The method according to claim 1further comprising administering to the individual an inhibitor of PGESand/or an antagonist of EP2 or EP4.
 13. The method according to claim 12wherein the antagonist of EP2 or EP4 is one or more of AH6809, anomega-substituted prostaglandin E derivative AH23848B, AH22921X,IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL, ILASYECL, IFTSTDCL, TSYEAL(with 4-biphenylalanine), TSYEAL (with homophenylalanine), a5-thia-prostaglandin E derivative5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,and5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.14. The method according to claim 1 further comprising administering tothe individual an agent that is an antagonist of the FP receptor. 15.The method according to claim 14 wherein the FP receptor antagonist isany one or more of PGF2α dimethyl amide; PGF2α dimethyl amine; AL-8810((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF2α); phloretin; glibenclamide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3(clseeakearrindeierqlrrdkrdarre-NH2); PCP-4 (kdtilqlnlkeynlv-NH2); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK); PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF). 16.The method according to claim 14 wherein the agent that is an antagonistof the FP receptor is an antagonist of PGF2α.
 17. The method accordingto claim 16 wherein the PGF2α antagonist is an anti-PGF2α antibody. 18.The method according to claim 1 further comprising administering to theindividual a cyclooxygenase-2 (COX-2) inhibitor.
 19. The methodaccording to claim 18 wherein the COX-2 inhibitor is any one ofnimesulide, 4-hydroxynimesulide, flosulide, and meloxicam. 20-42.(canceled)
 43. A composition comprising at least one agent that is anantagonist of the IP receptor and/or a PGIS inhibitor, and any one ormore of an inhibitor of PGES and/or an antagonist of EP2 or EP4, anagent that is an antagonist of the FP receptor, and a COX-2 inhibitor.44. (canceled)
 45. A pharmaceutical composition comprising thecomposition according to claim 43 and a pharmaceutically acceptablecarrier.
 46. A vaginal ring or a tampon or an intrauterine devicecomprising the composition according to claim
 43. 47-50. (canceled) 51.The method according to claim 1 further comprising administering to theindividual any one or more of an inhibitor of PGES and/or an antagonistof EP2 or EP4, an agent that is an antagonist of the FP receptor, and aCOX-2 inhibitor.
 52. The method according to claim 12 further comprisingadministering to the individual an agent that is an antagonist of the FPreceptor.
 53. The method according to claim 12 further comprisingadministering to the individual a COX-2 inhibitor.
 54. The methodaccording to claim 14 further comprising administering to the individuala COX-2 inhibitor.
 55. The method according to claim 1, wherein the atleast one agent is administered via a vaginal ring or a tampon or anintrauterine device.
 56. The composition according to claim 43, whereinthe antagonist of the IP receptor prevents or reduces the binding ofPGI2 to the IP receptor.
 57. The composition according to claim 43,wherein the antagonist of the IP receptor affects the interactionbetween PGI2 and the IP receptor, or the interaction between the IPreceptor and the associated G protein, thus inhibiting or disrupting aPGI2-IP mediated signal transduction pathway.
 58. The compositionaccording to claim 43, wherein the IP receptor antagonist is any one ormore of a 2-(arylphenyl)amino-imidazoline derivative; a2-(substituted-phenyl)amino-imidazoline derivative; analkoxycarbonylamino heteroaryl carboxylic acid derivative; analkoxycarbonylamino benzoic acid or alkoxycarbonylamino tetrazolylphenyl derivative; a 2-phenylaminoimidazoline phenyl ketone derivative;a carboxylic acid derivative; an amino- or amido-prostacyclin derivativecompound; a 15(R)-isocarbacyclin or 15-deoxyisocarbacyclin derivative; a6,9-thiaprostacyclin analogue or derivative; (5Z)-carbacyclin; FCE 22176((5Z)-13,14-didehydro-20-methyl-carboprostacyclin); and an anti-IPreceptor antibody.
 59. The composition according to claim 43, whereinthe agent is an antagonist of PGI2.
 60. The composition according toclaim 43, wherein the agent is an inhibitor of PGIS.
 61. The compositionaccording to claim 60, wherein the PGIS inhibitor is an anti-PGISantibody, U-51605, peryoxynitrite, 3-morpholinosydnonimineN-ethylcarbamide or trans-2-phenylcyclopropylamine HCl.
 62. Thecomposition according to claim 43, wherein the antagonist of EP2 or EP4is one or more of AH6809, an omega-substituted prostaglandin Ederivative, AH23848B, AH22921X, IFTSYLECL, IFASYECL, IFTSAECL, IFTSYEAL,ILASYECL, IFTSTDCL, TSYEAL (with 4-biphenylalanine), TSYEAL (withhomophenylalanine), a 5-thia-prostaglandin E derivative,5-butyl-2,4-dihydro-4-[[2′-[N-(3-chloro-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-onepotassium salt,5-butyl-2,4-dihydro-4-[[2′-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,5-butyl-2,4-dihydro-4-[[2′-[N-(2-thiophenecarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,and5-butyl-2,4-dihydro-4-[[2′-[N-[2-(methypyrrole)carbonyl]sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one.63. The composition according to claim 43, wherein the FP receptorantagonist is any one or more of PGF2a dimethyl amide; PGF2α dimethylamine; AL-8810 ((5Z,13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoicacid); AL-3138 (11-deoxy-16-fluoro PGF2α); phloretin; glibenclainide;ridogrel; PHG113; PCP-1 (rvkfksqqhrqgrshhlem); PCP-2(rkavlknlyklasqccgvhvislhiwelssiknslkvaaisespvaeksast); PCP-3(clseeakearrindeierqlrrdkrdarre-NH2); PCP-4 (kdtilqlnlkeynlv-NH2); PCP-8(ilghrdyk); PCP-10 (wedrfyll); PCP-13 (ILGHRDYK); PCP-14 (YQDRFYLL);(ILAHRDYK); PCP-13.7 (ILAHRDYK); PCP-13.8 (ILaHRDYK); PCP-13.11(ILGFRDYK); PCP-13.13 (ILGHKDYK); PCP-13.14 (ILGHRNYK); PCP-13.18(ILGHQDYK); PCP-13.20 (ILGHRDY-amide); PCP-13.21 (ILGHRDYK-amide);PCP-13.22 (ILGWRDYK); PCP-13.24 (ILGXRDYK); and PCP-15 (SNVLCSIF). 64.The composition according to claim 43, wherein the agent that is anantagonist of the FP receptor is an antagonist of PGF2α.
 65. Thecomposition according to claim 64, wherein the PGF2α antagonist is ananti-PGF2α antibody.
 66. The composition according to claim 43, whereinthe COX-2 inhibitor is any one of nimesulide, 4-hydroxynimesulide,flosulide, and meloxicam.